Methods and materials for biosynthesis of mogroside compounds

ABSTRACT

The invention relates to methods for producing mogrosides with the aid of enzymes. In particular the invention proposes various biosynthetic pathways useful for mogroside production and enzymes useful for mogroside production are provided. Furthermore, the invention provides recombinant hosts useful in performing the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. Ser. No. 13/442,694, filed May13, 2015, now U.S. Pat. No. 9,932,619, granted Apr. 3, 2018, which is aU.S. national phase of International Application No. PCT/EP2013/075510filed Dec. 4, 2013, which claims the benefit of U.S. ProvisionalApplication No. 61/733,220 filed Dec. 4, 2012. The entire disclosurecontents of these applications are herewith incorporated by reference intheir entirety into the present application.

FIELD OF INVENTION

The present invention relates to methods and materials for biosynthesisof mogroside compounds, and more particularly to methods involving useof cytochrome P450 enzymes to produce mogrol and/or usinguridine-5′-diphospho (UDP) dependent glucosyltransferases (UGTs) toglycosylate mogrol and produce various mogrol glycosides (mogrosides).The methods may also involve use of enzymes involved in biosynthesis ofsubstrates for mogrol production.

BACKGROUND OF INVENTION

Mogrosides are a family of triterpene glycosides isolated from fruits ofSiraitia grosvenorii (Swingle), also known as Momordica grosvenori(Swingle). Extracts of the fruits are commercially used as naturalsweeteners. Four major compounds, Mogroside V, Mogroside IV, SiamenosideI, and 11-Oxomogroside V, have been identified from the fruits ofSiraitia grosvenorii (Swingle) that are responsible for the sweetness ofthe fruits (see FIG. 1). Mogroside V is the most abundant of these fourcompounds at approximately 0.57% (w/w) of the dry fruit, followed byMogroside IV and Siamenoside I, each of which contain four glucosemoieties. 11-Oxomogroside V has a ketone group instead of a hydroxyl atC-11. See, e.g., Takemoto, et al., Yakugaku Zasshi, 103, 1151-1154;1155-1166; 1167-1173, (1983); Kasai, et al., Agric. Biol. Chem. 53,3347-3349 (1989); Matsumoto, Chem. Pharm. Bull. 38, 2030-2032 (1990);and Prakash, et al., J. Carbohydrate Chem. 30, 16-26 (2011). However,the enzymes responsible for producing mogrosides have not beenidentified.

Tang et al. BMC Genomics 2011, 12:343 describes seven CYP450s and fiveUDPGs as potential candidates involved in mogroside biosynthesis.However, the document does not specifically identify any CYPs or UDPGsinvolved in mogroside biosynthesis.

SUMMARY OF INVENTION

The present invention provides methods and materials for biosynthesis ofmogroside compounds. Interestingly, the invention provides enzymesinvolved in mogroside biosynthesis.

Mogroside biosynthesis may involve several steps, and accordingly it isan aspect of the present invention to provide enzymes capable ofcatalysing each of these steps. It is however also foreseen that themethods may involve performing only some of the steps enzymatically,whereas others may be performed by other means.

In one aspect, this document features a method of producing a mogrosidecompound.

Thus, the invention provides a method of producing a mogroside, whereinthe method comprises one or more of the following steps:

-   -   Step Ia. Enhancing levels of oxido-squalene    -   Step Ib. Enhancing levels of dioxido-squalene    -   Step IIa. Oxido-squalene→cucurbitadienol    -   Step IIb. Dioxido-squalene→24,25 epoxy cucurbitadienol    -   Step IIIa. Cucurbitadienol→11-hydroxy-cucurbitadienol    -   Step IIIb. 24,25 epoxy cucurbitadienol→11-hydroxy-24,25 epoxy        cucurbitadienol    -   Step IVa. 11-hydroxy-cucurbitadienol→mogrol    -   Step IVb. 11-hydroxy-24,25 epoxy cucurbitadienol→mogrol    -   Step V mogrol→mogroside

Methods for performing each of the above-mentioned steps are describedherein below. In particular, enzymes or mixture of enzymes useful foreach of above-mentioned steps are described in details herein below.

The invention also features a recombinant host comprising one or more ofthe following heterologous nucleic acids:

-   -   IIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIa        (Oxido-squalene→cucurbitadienol)    -   IIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIb        (Dioxido-squalene→24,25 epoxy cucurbitadienol)    -   IIIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIa        (Cucurbitadienol→11-hydroxy-cucurbitadienol)    -   IIIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIb (24,25 epoxy        cucurbitadienol→11-hydroxy-24,25 epoxy cucurbitadienol)    -   IVa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVa        (11-hydroxy-cucurbitadienol→mogrol)    -   IVb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVb (11-hydroxy-24,25        epoxy cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

In addition to the heterologous nucleic acids, said recombinant host mayhave been modified to achieve Step Ia and/or Step Ib.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting. Other featuresand advantages of the invention will be apparent from the followingdetailed description. Applicants reserve the right to alternativelyclaim any disclosed invention using the transitional phrase“comprising,” “consisting essentially of,” or “consisting of,” accordingto standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 contains the chemical structure of Mogroside V, Mogroside IV,Siamenoside I, and 11-Oxomogroside V.

FIG. 2 is a schematic of the pathway for the production of mogrosidesfrom glucose.

FIG. 3 is a schematic of the production of mogrol glycosides(mogrosides) from squalene.

FIG. 4 is a schematic of the pathway proposed herein by the inventors(top) and published (bottom) of a P450 pathway for formation of mogrolfrom cucurbitadienol.

FIG. 5 is a depiction of the biosynthesis of mogroside I E1, mogroside IA1, mogroside II E, mogroside III A2, mogroside III, mogroside IV, andmogroside V from mogrol using UGTs.

FIG. 6 is a schematic of the products obtained from mogroside V afterincubation with a pectinase and/or a cellulase.

FIG. 7 shows the LC-MS mass peak 501 corresponding to the proton plusNa+ adduct of tetrahydroxysqualene in a sample from yeast strainEFSC3027 transformed with a plasmid expressing S. grosvenorii Epoxidehydrolase 2.

FIG. 8 shows the LC-MS chromatogram peak of lanosterol in yeast strain(upper panel) and LC-MS chromatogram peaks of cucurbitadienol andlanosterol in yeast strain EFSC3498, which expresses cucurbitadienolsynthase (lower panel).

FIG. 9 shows the LC-MS chromatogram with the three peaks made whenCYP5491 and CPR4497 are expressed in yeast strain EFSC3498 (upperpanel), while the three lower panels show the fragmentation spectrum ofthese three peaks. The masses of the 3 peaks (443.38, 441.37 and 457.36)correspond in weight to proton adducts of hydroxylated cucurbitadienol,oxo cucurbitadienol and hydroxy plus oxo cucurbitadienol respectively.

FIG. 10A shows a route from oxido-squalene to mogrol and 11-oxo-mogrolproposed by the present invention.

FIG. 10B shows a route from dioxido-squalene to mogrol and 11-oxo-mogrolproposed by the present invention.

FIG. 11A shows the LC-MS chromatogram of reference mogroside I A1, whileFIG. 11B shows the LC-MS chromatogram of a sample of yeast strainEFSC1563 expressing UGT1576 in a culture fed 50 uM mogrol.

FIG. 12A shows the LC-MS chromatograms of samples from yeast strainEFSC1563 co-expressing UGT SK98 with UGT1576 showing production ofdi-glycosylated mogrol (mogroside II A). FIG. 12B shows LC-MSchromatograms of samples from yeast strain EFSC1563 co-expressing UGT98with UGT1576 showing production of di and tri-glycosylated mogrol(middle and lower frames).

FIG. 13 shows a route from mogrol to Mogroside III A1 proposed by thepresent invention.

FIG. 14 shows the amino acid sequence of a cucurbitadienol synthase fromCucurbita pepo (SEQ ID NO:1).

FIG. 15 shows the nucleic acid sequences of CYP533, CYP937, CYP1798,CYP1994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491,CYP5491, CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, CYP10285, andCYP10969 (SEQ ID NOs:3-20 and 41, respectively).

FIG. 16 shows the amino acid sequences of UGT73C3, UGT73C5, UGT73C6,UGT73E1, and UGT85C2 (SEQ ID NOs:21-25, respectively).

FIG. 17 shows the nucleic acid sequences of UGT98, UGT1495, UGT1817,UGT3494 (partial gene sequence), UGT5914, UGT8468, UGT10391, UGT11789(partial gene sequence), UGT11999 (partial gene sequence), UGT13679(partial gene sequence), and UGT15423 (partial gene sequence) (SEQ IDNOs:26-36, respectively).

DETAILED DESCRIPTION OF THE INVENTION

Method of Producing a Mogroside

This document is based on the invention that recombinant hosts such asmicroorganisms, plant cells, or plants can be developed that expresspolypeptides useful for the biosynthesis of mogrol (the triterpene core)and various mogrol glycosides (mogrosides). The aglycone mogrol isglycosylated with different numbers of glucose moieties to form variousmogroside compounds. Recombinant microorganisms are particularly usefulhosts. The recombinant host may be any of the recombinant hostsdescribed herein below in the section “Recombinant host”.

Expression of these biosynthetic polypeptides in various microbialchassis allows mogrol and its glycosides to be produced in a consistent,reproducible manner from energy and carbon sources such as sugars,glycerol, CO₂, H₂ and sunlight. FIG. 2 provides a schematic of thepathway for production of mogrol and various mogrosides from glucose.

It is one aspect of the invention to provide a method of producing amogroside, wherein the method comprises one or more of the followingsteps:

-   -   Step Ia. Enhancing levels of oxido-squalene    -   Step Ib. Enhancing levels of dioxido-squalene    -   Step IIa. Oxido-squalene→cucurbitadienol    -   Step IIb. Dioxido-squalene→24,25 epoxy cucurbitadienol    -   Step IIIa. Cucurbitadienol→11-hydroxy-cucurbitadienol    -   Step IIIb. 24,25 epoxy cucurbitadienol→11-hydroxy-24,25 epoxy        cucurbitadienol    -   Step IVa. 11-hydroxy-cucurbitadienol→mogrol    -   Step IVb. 11-hydroxy-24,25 epoxy cucurbitadienol→mogrol    -   Step V mogrol→mogroside

Methods and materials for performing each of the steps are described inmore detail herein below. Each of the steps of the method results ingeneration of a product. Said products may also be referred to as“intermediate products” herein. Each step uses a substrate, which mayalso be referred to as “precursor molecules”. It is clear from abovethat the intermediate products also may serve as precursor molecules fora subsequent step.

Thus, the invention provides methods of producing mogrosides, whereinthe method may comprise the steps of

-   -   Step Ia. Enhancing levels of oxido-squalene    -   Step IIa. Oxido-squalene→cucurbitadienol    -   Step IIIa. Cucurbitadienol→11-hydroxy-cucurbitadienol    -   Step IVa. 11-hydroxy-cucurbitadienol→mogrol    -   Step V. mogrol→mogroside

and optionally isolating said mogroside.

The invention also provides methods of producing mogrosides, wherein themethod may comprise the steps of

-   -   a) Providing oxido-squalene    -   b) Performing Steps IIa, IIIa, IVa and V identified above    -   c) optionally isolating said mogroside.

The invention also provides methods of producing mogrosides, wherein themethod may comprise the steps of

-   -   Step Ib. Enhancing levels of dioxido-squalene    -   Step IIb. Dioxido-squalene→24,25 epoxy cucurbitadienol    -   Step IIIb. 24,25 epoxy cucurbitadienol→11-hydroxy-24,25 epoxy    -   cucurbitadienol    -   Step IVb. 11-hydroxy-24,25 epoxy cucurbitadienol→mogrol    -   Step V. mogrol→mogroside

and optionally isolating said mogroside.

The invention also provides methods of producing mogrosides, wherein themethod may comprise the steps of

-   -   a) providing dioxido-squalene    -   b) performing steps IIb, IIIb, IVb and V identified above    -   c) optionally isolating said mogroside.

The invention also provides methods of producing mogrosides, whereinsaid mogroside may be a higher glycosylated mogroside, wherein themethod may comprise the steps of

-   -   a) providing cucurbitadienol    -   b) performing steps IIIa, IVa and V identified above    -   c) optionally isolating said mogroside.

The invention also provides methods of producing mogrosides, whereinsaid mogroside may be a higher glycosylated mogroside, wherein themethod may comprise the steps of

-   -   a) providing 24,25 epoxy cucurbitadienol    -   b) performing steps IIIb, IVb and V identified above    -   c) optionally isolating said mogroside.

The invention provides methods of producing mogrosides, wherein themethod may comprise the steps of

-   -   a) providing mogrol    -   b) performing step V identified above    -   c) optionally isolating said mogroside.

The invention provides methods of producing mogrol, wherein the methodmay comprise the steps of

-   -   a) providing dioxido-squalene    -   b) performing steps IIb, IIb and IVb identified above    -   c) optionally isolating said mogrol.

In general, the method may be performed either in vitro or in vivo. Itis also comprised within the invention that some steps are performed invitro, whereas others may be performed in vivo. Thus, for example thefirst steps may be performed in vitro and where after an intermediateproduct may be fed to recombinant host cells, capable of performing theremaining steps of the method. Alternatively, the first steps may beperformed in vivo and where after an intermediate product may be used assubstrate for the subsequent step(s) performed in vitro. Othercombinations can also be envisaged.

When said methods are performed in vitro each of the steps of themethods may be performed separately. Alternatively, one or more of thesteps may be performed within the same mixture. In embodiments whereinsome or all of the steps of the methods are performed separately, thenthe intermediate product of each of the steps may be purified or partlypurified before performing the next step.

When said methods are performed in vivo, the methods employ use of arecombinant host expressing one or more of said enzymes or the methodsmay employ use of several recombinant hosts expressing one or more ofsaid enzymes. The methods may also employ a mixture of recombinant andnon-recombinant host. If more than one host is used then the hosts maybe co-cultivated, or they may be cultured separately. If the hosts arecultivated separately the intermediate products may be recovered andoptionally purified and partially purified and fed to recombinant hostsusing the intermediate products as substrates. Useful recombinant hoststo be used with the invention are described herein below.

Said oxido-squalene, dioxido-squalene, cucurbitadienol, 24,25 epoxycucurbitadienol or mogrol may be provided in any suitable manner. Forexample said oxido-squalene, dioxido-squalene, cucurbitadienol, 24,25epoxy cucurbitadienol or mogrol may be provided in isolated form or aspart of a composition or an extract. In embodiments of the invention,wherein the methods are performed in vivo, said oxido-squalene,dioxido-squalene, cucurbitadienol, 24,25 epoxy cucurbitadienol or mogrolmay be added to the cultivation medium. It is also comprised within theinvention that a recombinant host is used, which endogenously expressesoxido-squalene, dioxido-squalene, cucurbitadienol, 24,25 epoxycucurbitadienol or mogrol.

Recombinant hosts described herein below can be used in methods toproduce mogroside compounds. For example, if the recombinant host is amicroorganism, the method can include growing the recombinantmicroorganism in a culture medium under conditions in which one or moreof the enzymes catalyzing step(s) of the methods of the invention, e.g.synthases, hydrolases, CYP450s and/or UGTs are expressed. Therecombinant microorganism may be grown in a fed batch or continuousprocess. Typically, the recombinant microorganism is grown in afermenter at a defined temperature(s) for a desired period of time.

A cell lysate can be prepared from the recombinant host expressing oneor more enzymes and be used to contact a substrate, such that mogrosidecompounds can be produced. For example, a cell lysate can be preparedfrom the recombinant host expressing one or more UGTs and used tocontact mogrol, such that mogroside compounds can be produced.

In some embodiments, mogroside compounds can be produced using wholecells that are fed raw materials that contain precursor molecules, e.g.,mogrol. The raw materials may be fed during cell growth or after cellgrowth. The whole cells may be in suspension or immobilized. The wholecells may be in fermentation broth or in a reaction buffer. In someembodiments a permeabilizing agent may be required for efficienttransfer of substrate into the cells.

Levels of products, substrates and intermediates can be determined byextracting samples from culture media for analysis according topublished methods. Mogroside compounds can be recovered from the cultureor culture medium using various techniques known in the art.

Recombinant Host

This document also feature recombinant hosts. As used herein, the termrecombinant host is intended to refer to a host, the genome of which hasbeen augmented by at least one incorporated DNA sequence. Saidincorporated DNA sequence may be a heterologous nucleic acid encodingone or more polypeptides. Such DNA sequences include but are not limitedto genes that are not naturally present, DNA sequences that are notnormally transcribed into RNA or translated into a protein(“expressed”), and other genes or DNA sequences which one desires tointroduce into the non-recombinant host. It will be appreciated thattypically the genome of a recombinant host described herein is augmentedthrough the stable introduction of one or more recombinant genes. Saidrecombinant gene may also be a heterologous nucleic acid encoding one ormore polypeptides. Generally, the introduced DNA or heterologous nucleicacid is not originally resident in the host that is the recipient of theDNA, but it is within the scope of the invention to isolate a DNAsegment from a given host, and to subsequently introduce one or moreadditional copies of that DNA into the same host, e.g., to enhanceproduction of the product of a gene or alter the expression pattern of agene. In some instances, the introduced DNA or heterologous nucleic acidwill modify or even replace an endogenous gene or DNA sequence by, e.g.,homologous recombination or site-directed mutagenesis.

In particular, the recombinant host according to the present inventioncomprises one or more of the following heterologous nucleic acids:

-   -   IIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIa        (Oxido-squalene→cucurbitadienol)    -   IIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIb        (Dioxido-squalene→24,25 epoxy cucurbitadienol)    -   IIIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIa        (Cucurbitadienol→11-hydroxy-cucurbitadienol)    -   IIIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIb (24,25 epoxy        cucurbitadienol→11-hydroxy-24,25 epoxy cucurbitadienol)    -   IVa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVa        (11-hydroxy-cucurbitadienol→mogrol) IVb. Heterologous nucleic        acid(s) encoding an enzyme or mixture of enzymes capable of        catalysing Step IVb (11-hydroxy-24,25 epoxy        cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

In addition to the heterologous nucleic acids, said recombinant host mayhave been modified to achieve Step Ia and/or Step Ib.

Enzymes capable of catalysing each of these steps are described hereinbelow in more detail.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   IIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIa        (Oxido-squalene→cucurbitadienol)    -   IIIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIa        (Cucurbitadienol→11-hydroxy-cucurbitadienol)    -   IVa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVa        (11-hydroxy-cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

and optionally said recombinant host may further have been modified toachieve Step Ia.

Said recombinant host cell is in particular useful in methods forproducing mogrosides.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   IIIa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIa        (Cucurbitadienol→11-hydroxy-cucurbitadienol)    -   IVa. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVa        (11-hydroxy-cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

Said recombinant host cell is in particular useful in methods forproducing mogrosides comprising a step of providing curcubutadienol.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   V Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

Said recombinant host cell is in particular useful in methods forproducing mogrosides comprising a step of providing mogrol.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   IIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIb        (Dioxido-squalene→24,25 epoxy cucurbitadienol)    -   IIIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIb (24,25 epoxy        cucurbitadienol→11-hydroxy-24,25 epoxy cucurbitadienol)    -   IVb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVb (11-hydroxy-24,25        epoxy cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

And optionally said recombinant host may have been modified to achieveStep Ib.

Said recombinant host cell is in particular useful in methods forproducing mogrosides.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   IIIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIb (24,25 epoxy        cucurbitadienol→11-hydroxy-24,25 epoxy cucurbitadienol)    -   IVb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVb (11-hydroxy-24,25        epoxy cucurbitadienol→mogrol)    -   V. Heterologous nucleic acid(s) encoding an enzyme or mixture of        enzymes capable of catalysing Step V (mogrol→mogroside)

Said recombinant host cell is in particular useful in methods forproducing mogrosides comprising a step of providing 24,25 epoxycucurbitadienol.

In one embodiment of the invention, the recombinant host according tothe present invention may comprise the following heterologous nucleicacids:

-   -   IIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIb        (Dioxido-squalene→24,25 epoxy cucurbitadienol)    -   IIIb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IIIb (24,25 epoxy        cucurbitadienol→11-hydroxy-24,25 epoxy cucurbitadienol)    -   IVb. Heterologous nucleic acid(s) encoding an enzyme or mixture        of enzymes capable of catalysing Step IVb (11-hydroxy-24,25        epoxy cucurbitadienol→mogrol)

and optionally said recombinant host may have been modified to achieveStep Ib.

Said recombinant host cell is in particular useful in methods forproducing mogrol.

Suitable recombinant hosts include microorganisms, plant cells, andplants.

Thus, in one embodiment, a recombinant host that produces a mogrosidecompound can include a recombinant gene encoding at least a first UGTselected from the group consisting of 73C3, 73C6, 85C2, 73C5, and 73E1,and a recombinant gene encoding at least a second UGT selected from thegroup consisting of UGT98, UGT1495, UGT1817, UGT5914, UGT8468 andUGT10391. For example, a recombinant host can include a recombinant geneencoding at least one UGT selected from 73C3, 73C6, 85C2, and 73E1; arecombinant gene encoding 73C5; and a recombinant gene encoding at leastone UGT selected from the group consisting of UGT98, UGT1495, UGT1817,UGT5914, UGT8468 and UGT10391. One or more of the following also can beincluded in a recombinant host: a recombinant gene encoding acucurbitadienol synthase (e.g., from Cucurbita pepo or monk fruit); arecombinant gene encoding a cytochrome P450 polypeptide selected fromthe group CYP533, CYP937, CYP1798, CYP1994, CYP2048, CYP2740, CYP3404,CYP3968, CYP4112, CYP4149, CYP4491, CYP5491, CYP6479, CYP7604, CYP8224,CYP8728, CYP10020, and CYP10285 (SEQ ID NOs:3-20, respectively); and arecombinant gene encoding a squalene synthase (e.g., from Gynostemmapentaphyllum or Arabidopsis thaliana). CYP5491 has previously also beenreferred to as CYP87.

At least one of the genes in the recombinant host is a recombinant gene,the particular recombinant gene(s) depending on the species or strainselected for use. Additional genes or biosynthetic modules can beincluded in order to increase yield of mogrol and mogrosides, improveefficiency with which energy and carbon sources are converted to mogroland mogrosides, and/or to enhance productivity from the cell culture orplant.

The recombinant host further can include a recombinant gene encoding acucurbitadienol synthase and/or a recombinant gene encoding a cytochromeP450 polypeptide (e.g., CYP533, CYP937, CYP1798, CYP1994, CYP2048,CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491, CYP5491, CYP6479,CYP7604, CYP8224, CYP8728, CYP10020, or CYP10285) and/or a recombinantgene encoding a squalene synthase.

It is also comprised within the invention that the recombinant host maybe modified in order to reduce glucanase activity, in particularglucanase activity, which may result in deglucosylation of mogrosides.Thus, the recombinant host may for example be modified to reduce of evenabolish exo-1,3-beta-Glucanase activity. In embodiments of the inventionwhen the recombinant host is yeast, this may be accomplished by deletionof the EXG1 gene and/or of the EXG2 gene, both of which are encoding anexo-1,3-beta-Glucanase.

The term “recombinant gene” refers to a gene or DNA sequence that isintroduced into a recipient host, regardless of whether the same or asimilar gene or DNA sequence may already be present in such a host. Theterm “heterologous nucleic acid” refers to a nucleic acid that isintroduced into a recipient host, wherein said host does notendogenously comprise said nucleic acid. “Introduced,” or “augmented” inthis context, is known in the art to mean introduced or augmented by thehand of man. Thus, a recombinant gene may be a DNA sequence from anotherspecies, or may be a DNA sequence that originated from or is present inthe same species, but has been incorporated into a host by recombinantmethods to form a recombinant host. It will be appreciated that arecombinant gene that is introduced into a host can be identical to aDNA sequence that is normally present in the host being transformed, andis introduced to provide one or more additional copies of the DNA tothereby permit overexpression or modified expression of the gene productof that DNA.

A recombinant gene encoding a polypeptide described herein comprises thecoding sequence for that polypeptide, operably linked in senseorientation to one or more regulatory regions suitable for expressingthe polypeptide. Because many microorganisms are capable of expressingmultiple gene products from a polycistronic mRNA, multiple polypeptidescan be expressed under the control of a single regulatory region forthose microorganisms, if desired. A coding sequence and a regulatoryregion are considered to be operably linked when the regulatory regionand coding sequence are positioned so that the regulatory region iseffective for regulating transcription or translation of the sequence.Typically, the translation initiation site of the translational readingframe of the coding sequence is positioned between one and about fiftynucleotides downstream of the regulatory region for a monocistronicgene. In many cases, the coding sequence for a polypeptide describedherein is identified in a species other than the recombinant host, i.e.,is a heterologous nucleic acid. Thus, if the recombinant host is amicroorganism, the coding sequence can be from other prokaryotic oreukaryotic microorganisms, from plants or from animals. In some case,however, the coding sequence is a sequence that is native to the hostand is being reintroduced into that organism. A native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. Such sequences may then also beconsidered heterologous nucleic acids. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also may include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). A regulatory region is operably linked to a codingsequence by positioning the regulatory region and the coding sequence sothat the regulatory region is effective for regulating transcription ortranslation of the sequence. For example, to operably link a codingsequence and a promoter sequence, the translation initiation site of thetranslational reading frame of the coding sequence is typicallypositioned between one and about fifty nucleotides downstream of thepromoter. A regulatory region can, however, be positioned at furtherdistance, for example as much as about 5,000 nucleotides upstream of thetranslation initiation site, or about 2,000 nucleotides upstream of thetranscription start site.

The choice of regulatory regions to be included depends upon severalfactors, including, but not limited to, efficiency, selectability,inducibility, desired expression level, and preferential expressionduring certain culture stages. It is a routine matter for one of skillin the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. It will be understood that more than one regulatoryregion may be present, e.g., introns, enhancers, upstream activationregions, transcription terminators, and inducible elements. One or moregenes can be combined in a recombinant nucleic acid construct in“modules” useful for a discrete aspect of mogroside production.Combining a plurality of genes in a module, particularly a polycistronicmodule, facilitates the use of the module in a variety of species. Inaddition to genes useful for mogroside production, a recombinantconstruct typically also contains an origin of replication, and one ormore selectable markers for maintenance of the construct in appropriatespecies.

It will be appreciated that because of the degeneracy of the geneticcode, a number of nucleic acids can encode a particular polypeptide;i.e., for many amino acids, there is more than one nucleotide tripletthat serves as the codon for the amino acid. Thus, codons in the codingsequence for a given polypeptide can be modified such that optimalexpression in a particular host is obtained, using appropriate codonbias tables for that host (e.g., microorganism). Nucleic acids may alsobe optimized to a GC-content preferable to a particular host, and/or toreduce the number of repeat sequences. As isolated nucleic acids, thesemodified sequences can exist as purified molecules and can beincorporated into a vector or a virus for use in constructing modulesfor recombinant nucleic acid constructs.

A number of prokaryotes and eukaryotes are suitable for use asrecombinant hosts with the present invention. Thus, the recombinant hostmay e.g. be selected from the group consisting of gram-negativebacteria, yeast and fungi. A species and strain selected for use as amogroside production strain is first analyzed to determine whichproduction genes are endogenous to the strain and which genes are notpresent. Genes for which an endogenous counterpart is not present in thestrain are assembled in one or more recombinant constructs, which arethen transformed into the strain in order to supply the missingfunction(s). Thus, it may be analysed which of steps IIa, IIIa, IVa andV are already performed by the host, and then said host may be modifiedby introduction of heterologous nucleic acids encoding enzymescatalyzing the remaining steps. Similarly, it may be analysed which ofsteps IIb, IIIb, IVb and V are already performed by the host, and thensaid host may be modified by introduction of heterologous nucleic acidsencoding enzymes catalyzing the remaining steps. As mentioned before therecombinant host may also be modified to increase levels ofoxido-squalene and/or dioxido-squalene.

Exemplary prokaryotic and eukaryotic species useful as recombinant withthe present invention are described in more detail below. However, itwill be appreciated that other species may be suitable. For example, therecombinant host may be in a genus selected from the group consisting ofAgaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia,Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia,Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces,Schizosaccharomyces, Sphaceloma, Xanthophyllomyces and Yarrowia.Exemplary species from such genera useful as recombinant hosts includeLentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium,Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis,Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomycesdendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis andYarrowia lipolytica. In some embodiments, a recombinant host may be amicroorganism, for example an Ascomycete such as Gibberella fujikuroi,Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, orSaccharomyces cerevisiae. In some embodiments, a recombinant host may bea microorganism for example a prokaryote such as Escherichia coli,Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will beappreciated that certain microorganisms can be used to screen and testgenes of interest in a high throughput manner, while othermicroorganisms with desired productivity or growth characteristics canbe used for large-scale production of mogroside compounds. Inparticular, food grade microorganisms may be useful for large-scaleproduction purposes.

Saccharomyces cerevisiae

As described above the recombinant host may for example be Saccharomycescerevisiae. Saccharomyces cerevisiae is a widely used chassis organismin synthetic biology, and can be used as the recombinant microorganismplatform. There are libraries of mutants, plasmids, detailed computermodels of metabolism and other information available for S. cerevisiae,allowing for rational design of various modules to enhance productyield. Methods are known for making recombinant microorganisms. The VG4strain of S. cerevisiae from Brochado et al. 2010 (Microb Cell Fact.9:84) may be particularly useful. VG4 has the genotype ofpdc1Δgdh1Δ↑GDH2. Another very useful strain of S. cerevisiae is BY4742described herein below in Example 9, or the yeast strain described inKirby, J et al in FEBS Journal 275 (2008) 1852-1859.

Aspergillus spp.

The recombinant host may also be a Aspergillus species such as A.oryzae, A. niger and A. sojae. Aspergillus spp, such as theaforementioned are widely used microorganisms in food production, andcan also be used as the recombinant microorganism platform. Nucleotidesequences are available for genomes of A. nidulans, A. fumigatus, A.oryzae, A. clavatus, A. flavus, A. niger, and A. ferrous, allowingrational design and modification of endogenous pathways to enhance fluxand increase product yield. Any of these may be used recombinant hosts.Metabolic models have been developed for Aspergillus, as well astranscriptomic studies and proteomics studies. A. niger is cultured forthe industrial production of a number of food ingredients such as citricacid and gluconic acid, and thus species such as A. niger are generallysuitable for the production of food ingredients.

Escherichia coli

The recombinant host may also be Escherichia coli, which is anotherwidely used platform organism in synthetic biology. Similar toSaccharomyces, there are libraries of mutants, plasmids, detailedcomputer models of metabolism and other information available for E.coli, allowing for rational design of various modules to enhance productyield. Methods similar to those described above for Saccharomyces can beused to make recombinant E. coli microorganisms.

Rhodobacter spp.

The recombinant host may also be Rhodobacter. Similar to E. coli, thereare libraries of mutants available as well as suitable plasmid vectors,allowing for rational design of various modules to enhance productyield. Methods similar to those described above for E. coli can be usedto make recombinant Rhodobacter microorganisms.

Physcomitrella spp.

The recombinant host may also be Physcomitrella mosses. Physcomitrellamosses, when grown in suspension culture, have characteristics similarto yeast or other fungal cultures. This genera is becoming an importanttype of cell for production of plant secondary metabolites, which can bedifficult to produce in other types of cells.

Step Ia—Enhancing Levels of Oxido-Squalene

As described herein above the methods of the invention may comprise astep of enhancing the levels of oxido-squalene. This is in particularrelevant in methods comprising step IIa, wherein step IIa is performedin vivo. Step Ia may in particular be performed by modifying therecombinant host to be used with the methods in a manner enhancing thelevels of oxido-squalene in said recombinant host. The invention alsorelates to recombinant hosts modified to enhance the levels ofoxido-squalene.

Thus, the methods may also comprise one or more steps leading toformation of oxido-squalene, in particular to the formation of2,3-oxido-squalene. Said steps are preferably performed prior to stepIIa described below, or simultaneously herewith. FIG. 3 provides aschematic of the pathway from squalene to mogrosides.

One step in the production of oxido-squalene may be production ofsqualene from farnesyl pyrophosphate. One enzyme that catalyzes theproduction of squalene from farnesyl pyrophosphate is squalene synthase(also referred to as squalene synthase). Said squalene synthase may beany enzyme classified under EC 2.5.1.21. The reaction is typicallythought to proceed using NADPH as a cosubstrate. Accordingly, the methodmay comprise a step of production of squalene from farnesylpyrophosphate catalyzed by a squalene synthase in the presence of NADPH.In embodiments of the invention wherein the methods are performed invivo, the recombinant host may thus comprise a heterologous nucleic acidencoding a squalene synthase. Some recombinant hosts may comprise anendogenous squalene synthase in which case the endogenous enzyme maysuffice. Endogenous squalene production pathways exist in yeastmetabolism, and accordingly, if the recombinant host is yeast, then saidstep may be endogenous to the recombinant host.

The squalene synthase may be any useful squalene synthase. For examplethe squalene synthase may be squalene synthase from Gynostemmapentaphyllum (protein accession number C4P9M2), another cucurbitaceaefamily plant. The squalene synthase may also be selected from the groupsconsisting of squalene synthase of Arabidopsis thaliana (proteinaccession number C4P9M3), Brassica napus, Citrus macrophylla, Euphorbiatirucalli (protein accession number B9WZW7), Glycine max, Glycyrrhizaglabra (protein accession number Q42760, Q42761), Glycrrhiza uralensis(protein accession number D6QX40, D6QX41, D6QX42, D6QX43, D6QX44,D6QX45, D6QX47, D6QX39, D6QX55, D6QX38, D6QX53, D6QX37, D6QX35, B5AID5,B5AID4, B5AID3, C7EDD0, C6KE07, C6KE08, C7EDC9), Lotusjaponicas (proteinaccession number Q84LE3), Medicago truncatula (protein accession numberQ8GSL6), Pisum sativum, Ricinus communis (protein accession numberB9RHC3), and Prunus mume and functional homologues of any of theaforementioned sharing at least at least 70%, such as at least 80%, forexample at least 90%, such as at least 95%, for example at least 98%sequence identity therewith. Increased copy numbers, heterologousnucleic acids encoding squalene synthase, or increased expression of thenative squalene synthase may improve levels of mogrosides produced in arecombinant host.

Another step in the production of oxido-squalene may be production ofoxido-squalene from squalene. One enzyme that catalyzes the productionof oxido-squalene from squalene is squalene epoxidase (also referred toas squalene monoxygenase). Said squalene epoxidase may be any enzymeclassified under EC 1.4.99.7. The reaction is typically thought toproceed using NADPH as a cosubstrate. Accordingly, the method maycomprise a step of production of oxido-squalene from squalene catalyzedby a squalene epoxidase in the presence of NADPH. In embodiments of theinvention wherein the methods are performed in vivo, the recombinanthost may thus comprise a heterologous nucleic acid encoding a squaleneepoxidase. Some recombinant hosts may comprise an endogenous squaleneepoxidase, in which case the endogenous enzyme may suffice. Endogenousoxido-squalene production pathways exist in yeast metabolism, andaccordingly, if the recombinant host is yeast, then said step may beendogenous to the recombinant host. However, in order to enhance thelevel of oxido-squalene it may never-the-less be advantageous to expressaddition squalene epoxidase.

The squalene epoxidase may be any useful squalene epoxidase. Thesqualene epoxidase may for example be squalene epoxidase from Gynostemmapentaphyllum (protein accession number C4P9M2), a cucurbitaceae familyplant. The squalene epoxidase may also be selected from the groupconsisting of squalene epoxidase of Arabidopsis thaliana (proteinaccession number Q9SM02, O65403, O65402, O65404, O81000, or Q9T064),Brassica napus (protein accession number O65727, O65726), Euphorbiatirucalli (protein accession number A7VJN1), Medicago truncatula(protein accession number Q8GSM8, Q8GSM9), Pisum sativum, and Ricinuscommunis (protein accession number B9R6V0, B9S7W5, B9S6Y2, B9T0Y3,B9S7T0, B9SX91) and functional homologues of any of the aforementionedsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith. Increased copy numbers, heterologous nucleic acids encodingsqualene epoxidase, or increased expression of the native squaleneepoxidase may improve levels of mogrosides produced in a recombinanthost.

The squalene epoxidase may also be the product of the ERG1 gene of S.cerevisiae. Thus, the squalene epoxidase may be a polypeptide of SEQ IDNO:54 or a functional homologues thereof sharing at least 70%, such asat least 80%, for example at least 90%, such as at least 95%, forexample at least 98% sequence identity therewith.

In one embodiment the recombinant host comprises a heterologous nucleicacid encoding a squalene epoxidase operably linked to sequence directinghigh expression of said squalene epoxidase in said host cell. Thus, thesqualene epoxidase may be endogenous to the recombinant host, but theexpression level may be increased by additional copies of nucleic acidsencoding the squalene epoxidase and/or by use of stronger promoters.

Oxido-squalene serves as a substrate for production of lanosterol. Thus,in one embodiment the level of oxido-squalene may be increased byreducing the activity of lanosterol synthase. In recombinant hostsexpressing an endogenous lanosterol synthase, this may be achieved bysubstituting the endogenous promoter directed expression of lanosterolsynthase with a weaker promoter directing expression of a lower level oflanosterol synthase. In yeast the ERG7 gene encodes lanosterol synthase.Thus, when the recombinant host is yeast, then the promoter of the ERG7gene may be substituted for another promoter, which directs a level ofexpression, which is lower than the endogenous expression level of ERG7.The lanosterol synthase may thus be the product of the ERG7 gene of S.cerevisiae, the sequence of which is provided herein as SEQ ID NO:55 ora functional homologues thereof sharing at least 70%, such as at least80%, for example at least 90%, such as at least 95%, for example atleast 98% sequence identity therewith.

Examples of useful weak promoters include the methionine-repressiblepromoter of the MET3 gene or the CUP1 cupper inducible promoter.Non-limiting examples of how to reduce the activity of lanosterolsynthase are described in Example 9 herein below or in Kirby et al.,2008 (vide supra) both of which are incorporated by reference herein.The sequence of S. cerevisiae lanosterol synthase is provided as SEQ IDNO:55. Thus, when the recombinant host is S. cerevisiae, then it ispreferred that the polypeptide of SEQ ID NO:55 is expressed at a lowerlevel than the level of said polypeptide in wild type S. cerevisiae.Similarly, when the recombinant host expresses a polypeptide similar tothe polypeptide of SEQ ID NO:55 (e.g. at least 70% identical to SEQ IDNO:55), then it is preferred that said polypeptide at least 70%identical to SEQ ID NO:55 is expressed at a lower level than the levelof said polypeptide in the wild type host.

In addition, expression of a truncated form of the enzyme3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) may also lead enhancedlevels of oxido-squalene. A useful truncated form of yeast HMG reductase(tHMG1) is described in Donald et al., 1997, Appl. Environ. Microbiol.63, 3341-3344.

Step Ib—Enhancing Levels of Dioxido-Squalene

As described herein above the methods of the invention may comprise astep of enhancing the levels of dioxido-squalene. This is in particularrelevant in methods comprising step IIb, wherein step IIb is performedin vivo. Step Ib may in particular be performed by modifying therecombinant host to be used with the methods in a manner enhancing thelevels of dioxido-squalene in said recombinant host. The invention alsorelates to recombinant hosts modified to enhance the levels ofdioxido-squalene.

Thus, the methods may also comprise one or more steps leading toenhanced levels of dioxido-squalene. Said steps are preferably performedprior to step IIb described below, or simultaneously herewith.

The present invention describes that the levels of dioxido-squalene inparticular may be enhanced by high expression of a squalene epoxidase.Said squalene epoxidase may be any of the squalene epoxidase describedherein above in the section “Step Ia—Enhancing levels ofoxido-squalene”. In particular, the squalene epoxidase may be theproduct of the ERG1 gene of S. cerevisiae. Thus, the squalene epoxidasemay be a polypeptide of SEQ ID NO:54 or a functional homologues thereofsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith. High expression level may be achieved by introducing aheterologous nucleic acid encoding a squalene epoxidase into the hostcell operably linked to sequence directing high expression of saidsqualene epoxidase in said host cell. Thus, the squalene epoxidase maybe endogenous to the recombinant host, but the expression level may beincreased by additional copies of nucleic acids encoding the squaleneepoxidase and/or by use of stronger promoters.

The levels of dioxido-squalene may also be enhanced by reducing theactivity of lanosterol synthase. The activity of lanosterol synthase maybe reduced by any of the methods described herein above in the section“Step Ia—Enhancing levels of oxido-squalene”.

The levels of dioxido-squalene may also be enhanced by expression of atruncated form of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase(tHMG1) may also lead enhanced levels of oxido-squalene. A usefultruncated form of yeast HMG reductase (tHMG1) is described in Donald etal., 1997, Appl. Environ. Microbiol. 63, 3341-3344.

Step IIa—Oxido-Squalene→Cucurbitadienol

As described herein above the methods of the invention may comprise astep of producing cucurbitadienol from oxido-squalene, and in particularfrom 2,3-oxido-squalene using an enzyme or mixture of enzymes capable ofcatalysing conversion of oxido-squalene to form cucurbitadienol. Theinvention also relates to recombinant hosts comprising a heterologousnucleic acid encoding an enzyme capable of catalysing conversion ofoxido-squalene to cucurbitadienol.

The step may be performed in vitro by incubating a compositioncomprising oxido-squalene with said enzyme or a mixture of enzymescapable of catalyzing conversion of oxido-squalene to formcucurbitadienol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme or a mixture of enzymescapable of catalyzing conversion of oxido-squalene to formcucurbitadienol. Said recombinant host may be capable of producingoxido-squalene, for example because the recombinant host expresses oneor more enzymes of the oxido-squalene biosynthesis pathway.

Alternatively, oxido-squalene may be provided to said recombinant hostfor example in the cultivation medium.

Said enzyme or mixture of enzyme capable of catalyzing conversion ofoxido-squalene to form cucurbitadienol preferably comprises or consistsof a cucurbitadienol synthase.

Said cucurbitadienol synthase may be any useful cucurbitadienolsynthase, for example a cucurbitadienol synthase, which has beenclassified as an oxidosqualene cyclase, such as the oxidosqualenecyclase described by Shibuya, Tetrahedron, Vol 60: pp. 6995-7003 (2004).

The amino acid sequence of a cucurbitadienol synthase from Cucurbitapepo is provided herein as SEQ ID NO:1 and also is provided in GenBank®under Protein Accession No. BAD34645.1. In one embodiment of theinvention the cucurbitadienol synthase is a polypeptide of SEQ ID NO:1or a functional homologue thereof sharing at least 70%, such as at least80%, for example at least 90%, such as at least 95%, for example atleast 98% sequence identity therewith.

As described in Example 5, the cucurbitadienol synthase from monk fruitwas identified and the sequence of the C-terminal portion of thepolypeptide determined. The amino acid sequence of the C-terminalportion of the monk fruit polypeptide is provided herein as SEQ ID NO:2.SEQ ID NO:2 is 97.5% identical to residues 515 to 764 of the C. pepopolypeptide set forth in SEQ ID NO:1. Thus, in one embodiment of theinvention the cucurbitadienol synthase is a polypeptide comprising theamino acid sequence set forth in SEQ ID NO:2.

In a preferred embodiment the cucurbitadienol synthase is thepolypeptide of SEQ ID NO:43 or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Other homologous proteins can be found of similar length and havingapproximately 70% homology or higher to SEQ ID NO:1. Such homologsinclude the polypeptides from Lotus japonicas (BAE53431), Populustrichocarpa (XP_002310905), Actaea racemosa (ADC84219), Betulaplatyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera(XP_002264289), Centella asiatica (AAS01524), Panax ginseng (BAA33460),and Betula platyphylla (BAB83086). The cucurbitadienol synthase may beany of the aforementioned or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Step IIb—Dioxido-Squalene→24,25 Epoxy Cucurbitadienol

As described herein above the methods of the invention may comprise astep of producing 24,25 epoxy cucurbitadienol from dioxido-squaleneusing an enzyme or mixture of enzymes capable of catalysing conversionof oxido-squalene to form cucurbitadienol. The invention also relates torecombinant hosts comprising a heterologous nucleic acid encoding anenzyme capable of catalysing conversion of dioxido-squalene to 24,25epoxy cucurbitadienol.

The step may be performed in vitro by incubating a compositioncomprising dioxido-squalene with said enzyme or a mixture of enzymescapable of catalyzing conversion of dioxido-squalene to form 24,25 epoxycucurbitadienol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme or a mixture of enzymescapable of catalyzing conversion of dioxido-squalene to 24,25 epoxycucurbitadienol. Said recombinant host may be capable of producingdioxido-squalene, for example because the recombinant host expresses oneor more enzymes of the dioxido-squalene biosynthesis pathway. However,it is preferred that said recombinant host has been modified to enhancelevels of dioxido-squalene in any of the manners described herein abovein the section “Step Ib Enhancing levels of dioxido-squalene”.Alternatively, dioxido-squalene may be provided to said recombinant hostfor example in the cultivation medium.

Said enzyme or mixture of enzyme capable of catalyzing conversion ofdioxido-squalene to 24,25 epoxy cucurbitadienol preferably comprises orconsists of a cucurbitadienol synthase.

Said cucurbitadienol synthase may be any useful cucurbitadienolsynthase, for example a cucurbitadienol synthase, which has beenclassified as an oxidosqualene cyclase, such as the oxidosqualenecyclase described by Shibuya, Tetrahedron, Vol 60: pp. 6995-7003 (2004).In one embodiment of the invention the cucurbitadienol synthase is apolypeptide of SEQ ID NO:1 or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

In a preferred embodiment the cucurbitadienol synthase is thepolypeptide of SEQ ID NO:43 or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Other homologous proteins can be found of similar length and havingapproximately 70% homology or higher to SEQ ID NO:1. Such homologsinclude the polypeptides from Lotus japonicas (BAE53431), Populustrichocarpa (XP_002310905), Actaea racemosa (ADC84219), Betulaplatyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera(XP_002264289), Centella asiatica (AAS01524), Panax ginseng (BAA33460),and Betula platyphylla (BAB83086). The cucurbitadienol synthase may beany of the aforementioned or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Step IIIa—Cucurbitadienol→11-Hydroxy-Cucurbitadienol

As described herein above the methods of the invention may comprise astep of producing 11-hydroxy-cucurbitadienol from cucurbitadienol usingan enzyme or a mixture of enzymes capable of catalysing hydroxylation ofcucurbitadienol to form 11-hydroxy-cucurbitadienol.

The step may be performed in vitro by incubating a compositioncomprising cucurbitadienol with said enzyme capable of catalyzinghydroxylation of cucurbitadienol to form 11-hydroxy-cucurbitadienol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme capable of catalyzinghydroxylation of cucurbitadienol to form 11-hydroxy-cucurbitadienol.Said recombinant host may be capable of producing cucurbitadienol, forexample because the recombinant host expresses one or more enzymes ofthe cucurbitadienol biosynthesis pathway. Alternatively, cucurbitadienolmay be provided to said recombinant host for example in the cultivationmedium.

Said enzyme capable of catalyzing hydroxylation of cucurbitadienol toform 11-hydroxy-cucurbitadienol preferably is selected from the group ofcytochrome P450 enzymes.

As indicated in Example 7, one or more of CYP533, CYP937, CYP1798,CYP1994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491,CYP5491, CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, and CYP10285(encoded by SEQ ID NOs: 3-20, respectively) can be used to producemogrol. eYAC technology can be used to assess activity of the cytochromeP450 enzymes as set forth in Example 8. Alternatively, an in vitroreaction can be used to assess the activity. Thus, in one embodiment ofthe invention at least one cytochrome P450 enzyme is selected from thegroup consisting of polypeptides encoding by the nucleic acid sequenceSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20 or a or a functional homologue thereofsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith at the amino acid level.

In a preferred embodiment of the invention the enzyme capable ofcatalyzing hydroxylation of cucurbitadienol to form11-hydroxy-cucurbitadienol is CYP5491. Thus, the enzyme catalyzinghydroxylation of cucurbitadienol to form 11-hydroxy-cucurbitadienol maybe a polypeptide of SEQ ID NO:44 or a functional homologue thereofsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith.

In one embodiment of the invention this step may be aided by at leastone CYP activator. This step of the methods of the invention may thuscomprise use of a cytochrome P450 enzyme as described above incombination with at least one CYP activator. Thus, the recombinant hostmay in addition to containing heterologous nucleic acids encoding thecytochrome P450 enzymes described herein above also contain aheterologous nucleic acid encoding a CYP activator. Said CYP activatormay be any useful CYP activator, for example it may be a polypeptide bea polypeptide of SEQ ID NO:46 or a functional homologue thereof sharingat least 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Step IIIb 24,25 Epoxy Cucurbitadienol→11-Hydroxy-24,25 EpoxyCucurbitadienol

As described herein above the methods of the invention may comprise astep of producing 11-hydroxy-24,25 epoxy cucurbitadienol from 24,25epoxy cucurbitadienol using an enzyme capable of catalysinghydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25epoxy cucurbitadienol.

The step may be performed in vitro by incubating a compositioncomprising 24,25 epoxy cucurbitadienol with said enzyme capable ofcatalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form11-hydroxy-24,25 epoxy cucurbitadienol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme capable of catalyzinghydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25epoxy cucurbitadienol. Said recombinant host may be capable of producing24,25 epoxy cucurbitadienol, for example because the recombinant hostexpresses one or more enzymes of the 24,25 epoxy cucurbitadienolbiosynthesis pathway, e.g. cucurbitadienol synthase. Alternatively,24,25 epoxy cucurbitadienol may be provided to said recombinant host forexample in the cultivation medium.

Said enzyme capable of catalyzing hydroxylation of cucurbitadienol toform 11-hydroxy-cucurbitadienol preferably is selected from the group ofcytochrome P450 enzymes.

In a preferred embodiment of the invention the enzyme capable ofcatalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form11-hydroxy-24,25 epoxy cucurbitadienol is CYP5491. Thus, the enzymecatalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form11-hydroxy-24,25 epoxy cucurbitadienol may be a polypeptide of SEQ IDNO:44 or a functional homologue thereof sharing at least 70%, such as atleast 80%, for example at least 90%, such as at least 95%, for exampleat least 98% sequence identity therewith.

In one embodiment of the invention this step may be aided by at leastone CYP activator. This step of the methods of the invention may thuscomprise use of a cytochrome P450 enzyme as described above incombination with at least one CYP activator. Thus, the recombinant hostmay in addition to containing heterologous nucleic acids encoding thecytochrome P450 enzymes described herein above also contain aheterologous nucleic acid encoding a CYP activator. Said CYP activatormay be any useful CYP activator, for example it may be a polypeptide bea polypeptide of SEQ ID NO:46 or a functional homologue thereof sharingat least 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

Step IVa—11-Hydroxy-Cucurbitadienol→Mogrol

As described herein above the methods of the invention may comprise astep of producing mogrol from 11-hydroxy-cucurbitadienol using an enzymeor a mixture of enzymes capable of catalysing conversion of11-hydroxy-cucurbitadienol to form mogrol.

The step may be performed in vitro by incubating a compositioncomprising 11-hydroxy-cucurbitadienol with said enzyme or mixture ofenzymes capable of catalyzing conversion of 11-hydroxy-cucurbitadienolto form mogrol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme or mixture of enzymescapable of catalyzing conversion of 11-hydroxy-cucurbitadienol to formmogrol. Said recombinant host may be capable of producing11-hydroxy-cucurbitadienol, for example because the recombinant hostexpresses one or more enzymes of the 11-hydroxy-cucurbitadienolbiosynthesis pathway. Alternatively, 11-hydroxy-cucurbitadienol may beprovided to said recombinant host for example in the cultivation medium.

Said enzyme or mixture of enzymes capable of catalyzing conversion of11-hydroxy-cucurbitadienol to form mogrol preferably comprises one ormore enzymes with together has CYP450 activity and epoxide hydrolaseactivity.

Enzymes with CYP450 include for example the polypeptides encoding by thenucleic acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or a or afunctional homologue thereof sharing at least 70%, such as at least 80%,for example at least 90%, such as at least 95%, for example at least 98%sequence identity therewith at the amino acid level.

Another enzyme with CYP450 activity is CYP5491. Thus, the enzyme withCYP450 activity may be a polypeptide of SEQ ID NO:44 or a functionalhomologue thereof sharing at least 70%, such as at least 80%, forexample at least 90%, such as at least 95%, for example at least 98%sequence identity therewith.

In one embodiment of the invention this step may be aided by at leastone CYP activator. This step of the methods of the invention may thuscomprise use of a cytochrome P450 enzyme as described above incombination with at least one CYP activator. Thus, the recombinant hostmay in addition to containing heterologous nucleic acids encoding thecytochrome P450 enzymes described herein above also contain aheterologous nucleic acid encoding a CYP activator. Said CYP activatormay be any useful CYP activator, for example it may be a polypeptide bea polypeptide of SEQ ID NO:46 or a functional homologue thereof sharingat least 70%, such as at least 80%, for example at least 90%, such as atleast 95%, for example at least 98% sequence identity therewith.

The enzyme having epoxide hydrolase activity may for example be anenzyme classified under EC 3.3._._. Said epoxide hydrolase preferablycatalyses the following reaction:epoxide+H₂O→glycol

Examples of enzymes with epoxide hydrolase activity includes S.grosvenorii Epoxide hydrolase 1 and S. grosvenorii Epoxide hydrolase 2.Thus, the enzyme with epoxide hydrolase activity may be selected fromthe group consisting of polypeptides of SEQ ID NO:38, SEQ ID NO:40 andfunctional homologue thereof sharing at least 70%, such as at least 80%,for example at least 90%, such as at least 95%, for example at least 98%sequence identity therewith.

Step IVa—11-Hydroxy-24,25 Epoxy Cucurbitadienol→Mogrol

As described herein above the methods of the invention may comprise astep of producing mogrol from 11-hydroxy-24,25 epoxy cucurbitadienolusing an enzyme or a mixture of enzymes capable of catalysing conversionof 11-hydroxy-24,25 epoxy cucurbitadienol to form mogrol.

The step may be performed in vitro by incubating a compositioncomprising 11-hydroxy-24,25 epoxy cucurbitadienol with said enzyme ormixture of enzymes capable of catalyzing conversion of 11-hydroxy-24,25epoxy cucurbitadienol to form mogrol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme or mixture of enzymescapable of catalyzing conversion of 11-hydroxy-24,25 epoxycucurbitadienol to form mogrol. Said recombinant host may be capable ofproducing 11-hydroxy-24,25 epoxy cucurbitadienol, for example becausethe recombinant host expresses one or more enzymes of the11-hydroxy-24,25 epoxy cucurbitadienol biosynthesis pathway.Alternatively, 11-hydroxy-24,25 epoxy cucurbitadienol may be provided tosaid recombinant host for example in the cultivation medium.

Said enzyme or mixture of enzymes capable of catalyzing conversion of11-hydroxy-24,25 epoxy cucurbitadienol to form mogrol preferablycomprises an enzyme with epoxide hydrolase activity.

The enzyme having epoxide hydrolase activity may for example be anenzyme classified under EC 3.3._._. Said epoxide hydrolase preferablycatalyses the following reaction:epoxide+H₂O→glycol

Examples of enzymes with epoxide hydrolase activity includes S.grosvenorii Epoxide hydrolase 1 and S. grosvenorii Epoxide hydrolase 2.Thus, the enzyme with epoxide hydrolase activity may be selected fromthe group consisting of polypeptides of SEQ ID NO:38, SEQ ID NO:40 andfunctional homologue thereof sharing at least 70%, such as at least 80%,for example at least 90%, such as at least 95%, for example at least 98%sequence identity therewith.

Step V—Mogrol→Mogroside

The methods of invention may involve a step of glycosylating mogrol toform mogroside. This step is in general accomplished with the aid of anenzyme or a mixture of enzymes capable of catalyzing glycosylation ofmogrol and/or of glycosylated mogrol.

The mogroside may be any of the mogrosides described herein below in thesection “Mogrosides”.

Step V may be performed in vitro by incubating a composition comprisingmogrol with said enzyme or a mixture of enzymes capable of catalyzingglycosylation of mogrol. The step may also be divided into separatesteps, wherein each step involves glycosylation of mogrol orglycosylated mogrol.

The step may also be performed in vivo in a recombinant host comprisingheterologous nucleic acid(s) encoding an enzyme or a mixture of enzymescapable of catalyzing glycosylation of mogrol and optionally also ofglycosylated mogrol. Said recombinant host may be capable of producingmogrol, for example because the recombinant host expresses one or moreenzymes of the mogrol biosynthesis pathway. Alternatively, mogrol may beprovided to said recombinant host for example in the cultivation medium.

Said enzyme or mixture of enzyme capable of catalyzing glycosylation ofmogrol preferably comprises a Uridine-5′-diphospho (UDP) dependentglucosyltransferase (UGT). In particular, it is preferred that step Vcomprises use of a UGT.

Thus, step V may include incubating mogrol with at least oneUridine-5′-diphospho (UDP) dependent glucosyltransferase (UGT) toproduce a mogroside compound (e.g., mogroside I E1, mogroside I A1,mogroside II E, mogroside III A2, mogroside III, mogroside IV, mogrosideV, or a mogroside compound glycosylated at C24-OH).

The UGT may for example be selected from the group consisting of 73C3,73C6, 85C2, 73C5, and 73E1. The UGT may also be UGT73C3 of SEQ ID NO:21,UGT73C6 of SEQ ID NO:23, UGT85C2 of SEQ ID NO:25, UGT73C5 of SEQ ID NO:22, UGT73E1 of SEQ ID NO:24 or a functional homologue of any of theaforementioned sharing at least 70%, such as at least 80%, for exampleat least 90%, such as at least 95%, for example at least 98% sequenceidentity therewith.

The UGT may also be selected from the group consisting of UGT98,UGT1495, UGT1817, UGT5914, UGT8468 and UGT10391. The UGT may also beUGT98 of SEQ ID NO:53, UGT1495 encoded by SEQ ID NO:27, UGT1817 encodedby SEQ ID NO:28, UGT5914 encoded by SEQ ID NO:30, UGT8468 encoded by SEQID NO:31 and UGT10391 encoded by SEQ ID NO:32 or a functional homologueof any of the aforementioned sharing at least 70%, such as at least 80%,for example at least 90%, such as at least 95%, for example at least 98%sequence identity therewith at the amino acid level.

When the methods are performed in vitro the UGTs can for example berecombinantly produced or can be in a cell lysate of a recombinant host.This document also features a method of producing a mogroside compound,wherein the method includes contacting mogrol with a cell lysateprepared from a recombinant host expressing a UGT to produce a mogrosidecompound (e.g., mogroside I E1, mogroside I A1, mogroside II E,mogroside III A2, mogroside III, mogroside IV, mogroside V, or amogroside compound glycosylated at C24-OH). The UGT can be any of theabove mentioned UGTs.

This document provides methods and materials for glycosylating mogrolusing one or more Uridine-5′-diphospho (UDP) dependentglucosyltransferases (UGTs). As indicated below, at least five UGTs havebeen identified that glycosylate the aglycone mogrol. Each of the UGTsidentified herein are in glycosyltransferase family I. Thus, in onepreferred embodiment the UGT is a UGT in glycosyltransferase family I.

UGTs 73C3, 73C6, 85C2 and 73E1 are capable of catalyzing glycosylationat the C24-OH position of mogrol or mogroside (UGT #2 in FIG. 4).Accordingly, in methods of the invention wherein the mogroside to beproduced comprises a glycosylation at the 024-OH position then at leastone UGT may be UGT73C3 of SEQ ID NO:21, UGT73C6 of SEQ ID NO:23, UGT85C2of SEQ ID NO:25 or UGT73E1 of SEQ ID NO:24 or a or a functionalhomologue of any of the aforementioned sharing at least 70%, such as atleast 80%, for example at least 90%, such as at least 95%, for exampleat least 98% sequence identity therewith.

UGT73C5 is capable of catalyzing glycosylation at both the 03-OH ofmogrol and mogroside (UGT #1 in FIG. 4) and C24-OH position (UGT #2).Accordingly, in methods of the invention wherein the mogroside to beproduced comprises a glycosylation at the C24-OH position and/or aglycosylation at the 03-OH position, then at least one UGT may beUGT73C5 of SEQ ID NO:22 or a functional homologue of any of theaforementioned sharing at least 70%, such as at least 80%, for exampleat least 90%, such as at least 95%, for example at least 98% sequenceidentity therewith.

UGTs 73C3, 73C5, and 73C6 are from Arabidopsis thaliana. UGT 73E1 and85C2 are from Stevia rebaudiana. The amino acid sequences of UGTs 73C3,73C5, 73C6, 73E1, and 85C2 are provided herein as SEQ ID NOs: 21-25,respectively). Thus, UGTs 73C3, 73C6, 85C2, or 73E1 can be used toproduce mogroside I E1 from mogrol, and UGT73C5 can be used to producemogroside I A1 from mogrol. Mogroside I E1 can be converted to mogrosideII E using UGT73C5. Mogroside I A1 can be converted to mogroside II Eusing UGTs 73C3, 73C6, 85C2, or 73E1.

In one preferred embodiment of the invention at least one UGT is UGT1576of SEQ ID NO:48 or a functional homologue of any of the aforementionedsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith. This is in particular the case in embodiments of theinvention, wherein the mogroside to be produced comprises aglycosylation at the 24-OH position, because UGT1576 is aglycosyltransferase with mogrol 24-OH UDP-glycosyltransferase activity.

In one preferred embodiment of the invention at least one UGT is UGT98of SEQ ID NO:53 or a functional homologue thereof sharing at least 70%,such as at least 80%, for example at least 90%, such as at least 95%,for example at least 98% sequence identity therewith. This is inparticular the case in embodiments of the invention, wherein themogroside to be produced comprises a 1,2 glucosylation and a 1,6glycosylation of the glucose at position C-24 to form mogroside III A1.

In one preferred embodiment of the invention at least one UGT is UGTSK98 of SEQ ID NO:50 or a functional homologue of any of theaforementioned sharing at least 70%, such as at least 80%, for exampleat least 90%, such as at least 95%, for example at least 98% sequenceidentity therewith. This is in particular the case in embodiments of theinvention, wherein the mogroside to be produced comprise a 1,2glycosylation of the glucose at position C-24 to form mogroside II A.

As shown in FIG. 4, three enzymatic glycosylations convert mogroside IIE into mogroside V or 11-Oxo-mogroside V. First, two glucoses areattached with 1,6-bonds to the two glucose molecules already present inmogroside II E. Second, another glucose is added to the C24-boundglucose, with a 1,2 bond. Mogroside IV is an intermediate in which the1,2-bound glucose is missing at the C24-bound glucose. In siamenosidethis glucose is present, but the 1,6-bound glucose at the C3-boundglucose is missing. 11-Oxo-mogroside V is identical to mogroside V, onlythe 11-OH is oxidized. One or more of the following UGTs can be used toconvert mogroside II E to mogroside IV, mogroside V, 11-oxo-mogroside V,and siamenoside I: UGT98, UGT1495, UGT1817, UGT3494, UGT5914, UGT8468,UGT10391, UGT11789, UGT11999, UGT13679 and UGT15423 (SEQ ID NOs: 26-36,respectively) or functional. For example, one or more of UGT98, UGT1495,UGT1817, UGT5914, UGT8468 and UGT10391 can be used to produce mogrosideIV, mogroside V, 11-oxo-mogroside V, or siamenoside I.

In one embodiment of the invention step V comprises one or more of thefollowing steps:

-   -   a) Glucosylation of mogrol at C24 to form mogroside I A1    -   b) 1,6 glucosylation of the C24 bound glucose of mogroside I A1        to form mogroside II A    -   c) 1,2 glucosylation of the C24 bound glucose of mogroside IIa        to form mogroside III A1    -   d) Glucosylation of mogroside III A1 at the C3 to form        siamenoside 1    -   e) 1,6 glucosylation of the C3 bound glucose of siamenoside 1 to        form mogroside V

These steps may each be catalyzed by a UGT capable of catalyzing saidstep. Thus, for example step a) may for example be catalyzed by UGT1576of SEQ ID NO:48 or a functional homologue of any of the aforementionedsharing at least 70%, such as at least 80%, for example at least 90%,such as at least 95%, for example at least 98% sequence identitytherewith. Step b) may for example be catalyzed by UGT98 of SEQ ID NO:53or a functional homologue thereof sharing at least 70%, such as at least80%, for example at least 90%, such as at least 95%, for example atleast 98% sequence identity therewith. Step c) may for example becatalyzed by UGT98 of SEQ ID NO:53, UGT SK98 of SEQ ID NO:50 or afunctional homologue of any of the aforementioned sharing at least 70%,such as at least 80%, for example at least 90%, such as at least 95%,for example at least 98% sequence identity therewith. Step d) may forexample be catalyzed by UGT73C5 of SEQ ID NO:22 or a functionalhomologue thereof sharing at least 70%, such as at least 80%, forexample at least 90%, such as at least 95%, for example at least 98%sequence identity therewith. Step e) may for example be catalyzed by UGTof the UGT91 family. For example step e9 may be catalyzed by UGT98 ofSEQ ID NO:53 or a functional homologue thereof sharing at least 70%,such as at least 80%, for example at least 90%, such as at least 95%,for example at least 98% sequence identity therewith.

Activity of the UGTs can be assessed in vitro. For example, an in vitroUGT reaction mixture can include UGT enzyme, 4× Tris buffer, substrate(250 μM), UDPglucose (750 μM) and 1% alkaline phosphatase, in a totalreaction volume of about 50 μl. The reactions can be performed insterilized 96 well plates, and incubated overnight at 30° C. After theincubation, 25 μL of DMSO can be added to each reaction and the reactionplates centrifuged for 5 min. Samples can be taken from each well,filtered, and then analyzed via LC-MS.

Production of Polypeptides

As described herein above, the methods of the invention may be performedin in vitro or in vivo. In embodiments of the invention where themethods are performed in vitro one or more of the enzymes to be used inthe methods may be prepared using any conventional method for producingpolypeptides.

Thus, enzymes, such as synthases, hydrolyases, UGTs and CYP450polypeptides described herein can be produced using any method. Forexample, enzymes, such as synthases, hydrolyases, UGT or CYP450polypeptides can be produced by chemical synthesis. Alternatively,enzymes, such as synthases, hydrolyases, UGT or CYP450 polypeptidesdescribed herein can be produced by standard recombinant technologyusing heterologous expression vectors encoding enzymes, such assynthases, hydrolyases, UGT or CYP450 polypeptides. Expression vectorscan be introduced into host cells (e.g., by transformation ortransfection) for expression of the encoded polypeptide, which thenoptionally can be purified or partly purified. Crude extracts comprisingthe enzymes may also be used with the methods of the invention.Expression systems that can be used for small or large scale productionof enzymes, such as synthases, hydrolyases, UGT and CYP450 polypeptidesinclude, without limitation, microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant DNA, such asbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules described herein, or yeast (e.g.,S. cerevisiae or S. pombe) transformed with recombinant yeast expressionvectors containing the nucleic acid molecules described herein. Usefulexpression systems also include insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing thenucleic acid molecules described herein, or plant cell systems infectedwith recombinant virus expression vectors (e.g., tobacco mosaic virus)or transformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the nucleic acid molecules described herein.Enzymes, such as synthases, hydrolyases, UGT or CYP450 polypeptides alsocan be produced using mammalian expression system harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., the metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter and the cytomegaloviruspromoter), along with the nucleic acids described herein. Enzymes, suchas synthases, hydrolyases, UGT or CYP450 polypeptides to be used withthe methods of the invention may have an N-terminal or C-terminal tag asdiscussed below.

This document also provides isolated nucleic acids encoding the enzymesto be used in each of steps Ia, Ib, IIa, IIb, IIIa, IIIb, Iva, IVb and Vdescribed herein above, such as synthases, hydrolyases, UGT or CYP450polypeptides. An “isolated nucleic acid” refers to a nucleic acid thatis separated from other nucleic acid molecules that are present in agenome, including nucleic acids that normally flank one or both sides ofthe nucleic acid in a genome. The term “isolated” as used herein withrespect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome. Thus, the isolated nucleic acid may becDNA encoding any of the enzymes to be used with the methods of theinvention.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., any paramyxovirus,retrovirus, lentivirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not considered an isolated nucleicacid.

In some embodiments, a nucleic acid sequence encoding an enzyme to beused with the methods of the invention, such as synthases, hydrolyases,UGT or CYP450 polypeptides can include a tag sequence that encodes a“tag” designed to facilitate subsequent manipulation (e.g., tofacilitate purification or detection), secretion, or localization of theencoded polypeptide. Tag sequences can be inserted in the nucleic acidsequence encoding the enzyme, such that the encoded tag is located ateither the carboxyl or amino terminus of the enzyme. Non-limitingexamples of encoded tags include green fluorescent protein (GFP),glutathione S transferase (GST), HIS tag, and Flag™ tag (Kodak, NewHaven, Conn.). Other examples of tags include a chloroplast transitpeptide, a mitochondrial transit peptide, an amyloplast peptide, signalpeptide, or a secretion tag.

Functional Homologs

Functional homologs of the polypeptides described above are alsosuitable for use in the methods and recombinant hosts described herein.A functional homolog is a polypeptide that has sequence similarity to areference polypeptide, and that carries out one or more of thebiochemical or physiological function(s) of the reference polypeptide.Thus, functional homologues of the enzymes described herein arepolypeptides that have sequence similarity to the reference enzyme, andwhich are capable of catalyzing the same step or part of a step of themethods of the invention as the reference enzyme.

In general it is preferred that functional homologues share at leastsome degree of sequence identity with the reference polypeptide. Thus,it is preferred that a functional homologues of any of the polypeptidesdescribed herein shares at least 70%, such as at least 75%, such as atleast 80%, for example at least 85%, for example at least 90%, such asat least 95%, for example at least 98% sequence identity therewith.

Amino acid sequence identity requires identical amino acid sequencesbetween two aligned sequences. Thus, a candidate sequence sharing 80%amino acid identity with a reference sequence, requires that, followingalignment, 80% of the amino acids in the candidate sequence areidentical to the corresponding amino acids in the reference sequence.Identity according to the present invention is determined by aid ofcomputer analysis, such as, without limitations, the ClustalW computeralignment program (Higgins D., Thompson J., Gibson T., Thompson J. D.,Higgins D. G., Gibson T. J., 1994. CLUSTAL W: improving the sensitivityof progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic AcidsRes. 22:4673-4680), and the default parameters suggested therein. TheClustalW software is available as a ClustalW WWW Service at the EuropeanBioinformatics Institute. Using this program with its default settings,the mature (bioactive) part of a query and a reference polypeptide arealigned. The number of fully conserved residues are counted and dividedby the length of the reference polypeptide. The sequence identity isdetermined over the entire length of the reference polypeptide.

A functional homolog and the reference polypeptide may be naturaloccurring polypeptides, and the sequence similarity may be due toconvergent or divergent evolutionary events. As such, functionalhomologs are sometimes designated in the literature as homologs, ororthologs, or paralogs. Variants of a naturally occurring functionalhomolog, such as polypeptides encoded by mutants of a wild type codingsequence, may themselves be functional homologs. Functional homologs canalso be created via site-directed mutagenesis of the coding sequence fora polypeptide, or by combining domains from the coding sequences fordifferent naturally-occurring polypeptides (“domain swapping”).Techniques for modifying genes encoding functional homologues of anenzyme to be used with the methods of the invention, such as synthases,hydrolyases, UGT or CYP450 polypeptides described herein are known andinclude, inter alia, directed evolution techniques, site-directedmutagenesis techniques and random mutagenesis techniques, and can beuseful to increase specific activity of a polypeptide, alter substratespecificity, alter expression levels, alter subcellular location, ormodify polypeptide:polypeptide interactions in a desired manner. Suchmodified polypeptides are considered functional homologs. The term“functional homolog” is sometimes applied to the nucleic acid thatencodes a functionally homologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofenzymes to be used with the methods of the invention, such as synthases,hydrolyases, UGT or CYP450 polypeptides. Sequence analysis can involveBLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databasesusing one of the sequences identified herein encoding an enzyme to beused with the methods of the invention, such as synthases, hydrolyases,UGT amino acid sequence as the reference sequence. Amino acid sequenceis, in some instances, deduced from the nucleotide sequence. Thosepolypeptides in the database that have greater than 40% sequenceidentity are candidates for further evaluation for suitability assynthases, hydrolyases, UGT or CYP450 polypeptide. Amino acid sequencesimilarity allows for conservative amino acid substitutions, such assubstitution of one hydrophobic residue for another or substitution ofone polar residue for another. If desired, manual inspection of suchcandidates can be carried out in order to narrow the number ofcandidates to be further evaluated. Manual inspection can be performedby selecting those candidates that appear to have domains present inenzymes to be used with the methods of the invention, such as synthases,hydrolyases, UGT or CYP450 polypeptides, e.g., conserved functionaldomains. Conserved regions can be identified by locating a region withinthe primary amino acid sequence of a polypeptide that is a repeatedsequence, forms some secondary structure (e.g., helices and betasheets), establishes positively or negatively charged domains, orrepresents a protein motif or domain. See, e.g., the Pfam web sitedescribing consensus sequences for a variety of protein motifs anddomains on the World Wide Web at sanger.ac.uk/Software/Pfam/ andpfam.janelia.org/. The information included at the Pfam database isdescribed in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998);Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al.,Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can bedetermined by aligning sequences of the same or related polypeptidesfrom closely related species. Closely related species preferably arefrom the same family. In some embodiments, alignment of sequences fromtwo different species is adequate. Typically, polypeptides that exhibitat least about 40% amino acid sequence identity are useful to identifyconserved regions. Conserved regions of related polypeptides exhibit atleast 45% amino acid sequence identity (e.g., at least 50%, at least60%, at least 70%, at least 80%, or at least 90% amino acid sequenceidentity). In some embodiments, a conserved region exhibits at least92%, 94%, 96%, 98%, or 99% amino acid sequence identity. Sequenceidentity can be determined as set forth above.

Mogrosides

The present invention relates to methods for producing mogrosides andmaterials for use in such methods. The term “mogroside” as used hereinrefers to mogrol glycosylated at one or more positions. In particular,mogrosides according to the present invention may be mogrol glycosylatedwith one or more glucose residues at the positions 3 and/or 24. It isless preferred that mogrosides are glycosylated at the 11 and 25positions. Mogrol is a compound of formula I provided below, whereinboth R₁ and R₂ are —H.

It is preferred that the mogroside is a compound of the followingformula I:

wherein R₁ and R₂ independently are —H, mono-glucoside, di-glucoside,tri-glucoside, and at least one of R₁ and R₂ is not —H.

In particular the mogroside may be one the mogrosides described in Table1 herein below.

TABLE 1 Mogrosides of formula I Name R₁ R₂ mogroside V Glc6-Glc-Glc6-Glc2-Glc siamenoside I Glc- Glc6-Glc2-Glc- mogroside IV Glc6-Glc-Glc2-Glc- mogroside IV A Glc6-Glc- Glc6-Glc- mogroside III Glc-Glc6-Glc- mogroside III A1 H Glc6-Glc2-Glc- mogroside III A2 Glc6-Glc-Glc- mogroside III E Glc- Glc2-Glc- mogroside II A H Glc2-Glc- mogrosideII A1 H Glc6-Glc- mogroside II A2 Glc6-Glc- H mogroside II E Glc- Glc-mogroside I A1 H Glc- mogroside I E1 Glc- H Glc = glucose

Mogroside I A1 may sometimes be referred to as mogroside Ib. Mogroside IE1 may sometimes be referred to as mogroside Ia. Mogroside II E maysometimes be referred to as mogroside II. Mogroside III A2 may sometimesbe referred to as mogroside IIIa.

Mogroside III may sometimes be referred to as mogroside IIIb. Thisalternative nomenclature is for example used in U.S. Ser. No.61/733,220.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

SEQUENCE LISTING

SEQ ID NO: 1 Amino acid sequence of C. pepo cucurbitadienol synthase SEQID NO: 2 Amino acid sequence of C-terminal portion of Siraitiagrosvenorii cucurbitadienol synthase SEQ ID NO: 3 DNA sequence encodingCYP533 (nucleotide sequence of CYP533 gene) SEQ ID NO: 4 DNA sequenceencoding CYP937 (nucleotide sequence of CYP937 gene) SEQ ID NO: 5 DNAsequence encoding CYP1798 (nucleotide sequence of CYP1798 gene) SEQ IDNO: 6 DNA sequence encoding CYP1994 (nucleotide sequence of CYP1994gene) SEQ ID NO: 7 DNA sequence encoding CYP2048 (nucleotide sequence ofCYP2048 gene) SEQ ID NO: 8 DNA sequence encoding CYP2740 (nucleotidesequence of CYP2740 gene) SEQ ID NO: 9 DNA sequence encoding CYP3404(nucleotide sequence of CYP3404 gene) SEQ ID NO: 10 DNA sequenceencoding CYP3968 (nucleotide sequence of CYP3968 gene) SEQ ID NO: 11 DNAsequence encoding CYP4112 (nucleotide sequence of CYP4112 gene) SEQ IDNO: 12 DNA sequence encoding CYP4149 (nucleotide sequence of CYP4149gene) SEQ ID NO: 13 DNA sequence encoding CYP4491 (nucleotide sequenceof CYP4491 gene) SEQ ID NO: 14 DNA sequence encoding CYP5491 (nucleotidesequence of CYP5491 gene) SEQ ID NO: 15 DNA sequence encoding CYP6479(nucleotide sequence of CYP6479 gene) SEQ ID NO: 16 DNA sequenceencoding CYP7604 (nucleotide sequence of CYP7604 gene) SEQ ID NO: 17 DNAsequence encoding CYP8224 (nucleotide sequence of CYP8224 gene) SEQ IDNO: 18 DNA sequence encoding CYP8728 (nucleotide sequence of CYP8728gene) SEQ ID NO: 19 DNA sequence encoding CYP10020 (nucleotide sequenceof CYP10020 gene) SEQ ID NO: 20 DNA sequence encoding CYP10285(nucleotide sequence of CYP10285 gene) SEQ ID NO: 21 Amino acid sequenceof UGT73C3 SEQ ID NO: 22 Amino acid sequence of UGT73C5 SEQ ID NO: 23Amino acid sequence of UGT73C6 SEQ ID NO: 24 Amino acid sequence ofUGT73E1 SEQ ID NO: 25 Amino acid sequence of UGT85C2 SEQ ID NO: 26Nucleotide sequence encoding Siraitia grosvenorii UGT98 SEQ ID NO: 27Nucleotide sequence encoding Siraitia grosvenorii UGT1495 SEQ ID NO: 28Nucleotide sequence encoding Siraitia grosvenorii UGT1817 SEQ ID NO: 29Partial gene sequence - nucleotide sequence encoding fragment ofSiraitia grosvenorii UGT3494 SEQ ID NO: 30 Nucleotide sequence encodingSiraitia grosvenorii UGT5914 SEQ ID NO: 31 Nucleotide sequence encodingSiraitia grosvenorii UGT8468 SEQ ID NO: 32 Nucleotide sequence encodingSiraitia grosvenorii UGT10391 SEQ ID NO: 33 Partial gene sequence -nucleotide sequence encoding fragment of Siraitia grosvenorii UGT11789SEQ ID NO: 34 Partial gene sequence - nucleotide sequence encodingfragment of Siraitia grosvenorii UGT11999 SEQ ID NO: 35 Partial genesequence - Nucleotide sequence encoding fragment of Siraitia grosvenoriiUGT13679 SEQ ID NO: 36 Partial gene sequence - Nucleotide sequenceencoding fragment of Siraitia grosvenorii UGT15423 SEQ ID NO: 37 DNAsequence encoding S. grosvenorii Epoxide hydrolase 1 codon optimised forexpression in S. cerevisiae SEQ ID NO: 38 Amino acid sequence of S.grosvenorii Epoxide hydrolase 1 SEQ ID NO: 39 DNA sequence encoding S.grosvenorii Epoxide hydrolase 2 codon optimised for expression in S.cerevisiae SEQ ID NO: 40 Amino acid sequence of S. grosvenorii Epoxidehydrolase 2 SEQ ID NO: 41 DNA sequence encoding CYP10969 (nucleotidesequence of CYP10969 gene) SEQ ID NO: 42 DNA sequence encoding Siraitiagrosvenorii cucurbitadienol synthase codon optimized for expression inS. cerevisiae SEQ ID NO: 43 Amino acid sequence of Siraitia grosvenoriicucurbitadienol synthase SEQ ID NO: 44 Amino acid sequence of S.grosvenorii CYP5491 SEQ ID NO: 45 DNA sequence encoding S. grosvenoriiCPR4497 SEQ ID NO: 46 Amino acid sequence of S. grosvenorii CPR4497 SEQID NO: 47 DNA sequence encoding S. grosvenorii UGT1576 SEQ ID NO: 48Amino acid sequence of S. grosvenorii UGT1576 SEQ ID NO: 49 DNA sequenceencoding S. grosvenorii UGT SK98 SEQ ID NO: 50 Amino acid sequence of S.grosvenorii UGT SK98 SEQ ID NO: 51 DNA sequence encoding S. grosvenoriiUGT98 SEQ ID NO: 52 DNA sequence encoding S. grosvenorii UGT98 codonoptimised for expression in S. cerevisiae SEQ ID NO: 53 Amino acidsequence of S. grosvenorii UGT98 SEQ ID NO: 54 Amino acid sequence of S.cerevisiae squalene epoxidase encoded by the ERG1 gene SEQ ID NO: 55Amino acid sequence of S. cerevisiae lanosterol synthase encoded by theERG7 gene

EXAMPLES Example 1—Purification of Mogroside V

Mogroside V was purified from commercially available monk fruit extracts(PureLo®, Swanson) as follows. Three bottles of PureLo® (240 grams) weredissolved in water (900 mL), then loaded on a column of HP-20 resin (400gram resin). The column was washed with water (2.5 liters); then furtherwashed with 20% methanol-water. The product was eluted with methanol.After evaporation of solvents and drying under high vacuum, mogroside V(2.5 grams, ˜80% purity, 11-oxomogroside V was the major impurity) wasobtained.

Example 2—Enzymatic Synthesis of Mogrol from Mogroside V

Mogroside V (300 mg) was dissolved in 0.1M sodium acetate buffer (pH4.5, 100 mL), and crude pectinase from Aspergillus niger (25 mL, SigmaP2736) was added. The mixture was stirred at 50° C. for 48 hours. Thereaction mixture was extracted with ethyl acetate (2×100 ml). Theorganic extract was dried under vacuum then purified with preparativeHPLC. Pure mogrol (40 mg) was obtained and its structure confirmed byNMR and mass spectroscopy. See FIG. 6.

Example 3—Enzymatic Synthesis of Mogrol 3-O-glucoside (mogroside I E1)and Mogrol 24-O-glucoside (Mogroside I A1) from Mogroside V

Mogroside V (300 mg) was dissolved in 0.1M sodium acetate buffer (pH4.5, 100 ml), and crude pectinase from Aspergillus niger (25 ml, SigmaP2736) was added. The mixture was stirred at 50° C. for 6.5 hours. Thereaction mixture was extracted with ethyl acetate (2×100 ml). Theorganic extract was dried under vacuum then purified with preparativeHPLC. Pure mogroside I E1 (11.0 mg) and mogroside I A1 (8.0 mg) wereobtained. Their structures were confirmed by NMR and mass spectroscopy.See FIG. 6.

Example 4—In Vitro UGT Screening and Reactions

In vitro reactions of mogrol with a panel of 230 UGT enzymes wereperformed and the products were analyzed with LC-MS. The in vitro UGTreaction mixtures included 4× Tris buffer, mogrol (250 μM), UDP-glucose(750 μM) and 1% alkaline phosphatase. Five μl of each partially purifiedUGT enzyme or crude enzyme extract was added to the reaction, and thereaction volume brought to 50 μl with water. The reactions wereincubated overnight at 30° C. and performed in sterilized 96 wellplates. After the incubation, 25 μL of DMSO were added into eachreaction and the reaction plates were centrifuged for 5 min. Forty μLsamples were taken from each well and filtered, and were used for LC-MSanalysis. UGTs 73C3, 73C6 and 85C2 were found to convert all the mogrolsubstrate to mogroside I A1. UGT 73C5 makes both mogroside I E1 and IA1. In the reaction with UGT 20 73E1, although the reaction was notcomplete, mogroside I A1 was found as the major product, together with anew glycosylated mogrol (neither mogroside I E1 nor I A1; exact massshown as a mogroside I, presumably caused by a glycosylation event onC11-OH).

Example 5 Identifying the Monk Fruit Cucurbitadienol Synthase

The gene in monk fruit that codes for cucurbitadienol synthase is CirCS,and the partial gene sequence covering 338 of the supposedly 764 aminoacids was identified by doing a tBLASTn analysis of the assembled datawith a query cucurbitadienol synthase from Cucurbita pepo (accessionnumber BAD34645.1, SEQ ID NO:1). The partial CirCS is 97.5% identical tothe C. pepo gene at the protein level (SEQ ID NO:2; from residues 515 to764 of SEQ ID NO:1).

Example 6 Identifying Monk Fruit Candidate Genes for P450 EnzymesCatalyzing Formation of Mogrol from Cucurbitadienol

A pathway from cucurbitadienol to mogrol has been proposed by Tang etal., BMC Genomics, 12, 343 (2011). The intermediates cucurbitadienol andmogrol exist in the fruit as they have been isolated as minor products.See Ukiya, et al., J. Agric. Food Chem. 50, 6710-6715 (2002). Glycosideintermediates exist in both 11-hydroxy and 11-oxo series, and graduallychange from mogroside I to mogroside V as fruits ripen, which indicatesthat the triterpene core is fully oxidized by P450 enzymes before thesubsequent glycosylations. According to the scheme proposed by Tang etal., three independent cytochrome P450 enzyme-catalyzed oxidationsresults in mogrol formation from cucurbitadienol (lower route in FIG.4). The present inventors have found that the proposed primary reactionis highly unlikely. It is therefore submitted that the route may involveepoxidation by one cytochrome P450 enzyme, followed by a spontaneous orenzyme catalyzed hydration, and another P450 enzyme-catalyzed oxidation(visualized in the upper route in FIG. 4), or the route may comprisesimilar steps in another order as shown in FIG. 10A. The presentinventors also propose another route starting from dioxido-squalene,which is shown in FIG. 10B.

To identify the most likely candidate P450 genes from monk fruit, aBLAST database was made consisting of the polypeptide sequences of the239 public domain Arabidopsis thaliana cytochrome P450 enzymes,representing most known enzyme subfamilies and variations. The sequenceswere used in a tBLASTn (translated nucleotide database) analysis of theassembled monk fruit transcriptome data to identify all sequences with ahomology to any of the database query sequences with an E value of10E-10 or lower. Seventy-two sequences were identified. Typically, theability to assemble full or long gene lengths of expressed sequence tagsin a transcriptome study means that many sequence tags of the gene inquestion were present. In the current experiment, this indicates thatthe gene was highly expressed in the monk fruit tissue and thus has ahigh probability of being a candidate for one of the two P450 enzymes ofinterest. Of the 72 sequences, 18 were full length or almost fulllength. The assembled genes were designated CYP533, CYP937, CYP1798,CYP1994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491,CYP5491, CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, and CYP10285.

These are candidate genes for two P450 enzymes involved in catalyzingconversion of cucurbitadienol into mogrol. Full length gene sequenceswere amplified by PCR for the gene contigs CYP533, CYP937, CYP1798,CYP1994, CYP2740, CYP4112, CYP4149, CYP4491, CYP5491, CYP7604, CYP8224,and CYP10285, using monk fruit leaf genomic DNA or root cDNA andsequence overlap extension technology to remove resident intronsequences. The nucleotide sequences of CYP533, CYP937, CYP1798, CYP1994,CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491, CYP5491,CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, and CYP10285 are providedas SEQ ID NOs: 3-20, respectively.

Example 7—Identifying Monk Fruit Candidate Genes for GlycosyltransferaseEnzymes Catalyzing Formation of Mogroside V, 11-Oxo-Mogroside V,Mogroside IV, Mogrosides III A2 and b and Siamenoside from Mogroside IIE

Three enzymatic glycosylations are needed to convert mogroside II E intomogroside V or 11-Oxo-mogroside V. Two glucoses are attached with1,6-bonds to the two glucose molecules already present in mogroside IIE. This may be done by one UGT enzyme. Another glucose is added to theC24-bound glucose, with a 1,2 bond. Mogroside IV is an intermediate inwhich the 1,6-bound glucose is missing at the C24-bound glucose. Insiamenoside this glucose is present, but the 1,6-bound glucose at theC3-bound glucose is missing. 11-Oxo-mogroside V is identical tomogroside V, only the 11-OH is oxidized. See, FIG. 1 for the structuresof mogroside IV, mogroside V, 11-Oxo-mogroside V, and siamenoside.

To identify all possible UGT genes in the assembled monk fruittranscriptome data, a database was assembled consisting of thepolypeptide sequences of glycosyltransferases (UGTs) of all knownsub-families, a total of 160 sequences. A tBLASTn analysis was performedbetween this database and the assembled monk fruit data. UGTs performingdi-glycosylation (i.e., attaching a sugar to another sugar which in turnresides on an aglycon) invariably come from Family 1 UGT sub-families76, 79, 91 or 94 (with the latter three forming the “orthology group8”). While sub-family 76 enzymes usually make 1,3 bonds, orthology group8 UGTs always make 1,2 or 1,6 bonds.

Sequences were identified that showed more homology to orthology group 8enzymes than to any other UGT enzymes or any non-UGT genes. Thus 11contigs were identified as likely candidates to encode the twoglycosyltransferase genes needed to turn mogroside II E into mogrosideV: UGT98, UGT1495, UGT1817, UGT3494, UGT5914, UGT8468, UGT10391,UGT11789, UGT11999, UGT13679 and UGT15423 (SEQ ID NOs: 26-36,respectively). Of these we were able to amplify by PCR UGT98, UGT1495,UGT1817, UGT5914, UGT8468 and UGT10391, using monk fruit leaf genomicDNA or root cDNA. The amplified genes were inserted into E. coliexpression plasmid vectors.

The enzymes are expressed and purified on nickel columns. In vitroreactions of mogroside I A1, I E1 and II E with the panel of 6 purifiedUGT enzymes are performed and the products analyzed with LC-MS. The invitro UGT reaction mixtures include 4× Tris buffer, substrate (250 μM),UDP-glucose (750 μM) and 1% alkaline phosphatase.

Five μl of each partially purified UGT enzyme are added to the reaction,and the reaction volume brought to 50 μl with water. The reactions areincubated overnight at 30° C. and performed in sterilized 96 wellplates. After the incubation, 25 μL of DMSO are added into each reactionand the reaction plates are centrifuged for 5 min. Forty μL samples aretaken from each well and filtered, and then analyzed via LC-MS. The UGTcatalyzing the 1,6-bond formation as well as the enzyme catalyzing the1,2-bond formation are identified based on the LC-MS analysis.

Example 8 Using eYAC Technology to Identify the Cytochrome P450 EnzymesResponsible for Turning Cucurbitadienol into Mogrol

eYAC gene expression technology was used to identify the activecytochrome P450 enzymes within a collection of candidate genes. Thefollowing genes were inserted into “Entry vectors” (a collection ofplasmid vectors containing gene promoter and terminator sequences whichhave different nucleotide sequence but which are all 30 repressible bythe addition of the amino acid methionine): the Cucurbita pepocucurbitadienol synthase gene, CYP533 (SEQ ID NO:3), CYP937 (SEQ IDNO:4), CYP1798 (SEQ ID NO:5), CYP1994 (SEQ ID NO:6), CYP2740 (SEQ IDNO:8), CYP4112 (SEQ ID NO:11), CYP4149 (SEQ ID NO:12), CYP4491 (SEQ IDNO:13), CYP5491 (SEQ ID NO:14), CYP7604 (SEQ ID NO:16), CYP8224 (SEQ IDNO:17), and CYP10285 (SEQ ID NO:20), the two cytochrome P450oxidoreductase (CPR) genes from Arabidopsis thaliana (ATR1 and ATR2), aCPR from Stevia rebaudiana (CPR8), a CPR isolated from monk fruit, andthe glycosyltransferases UGT73C5 (SEQ ID NO: 22) and UGT73C6 (SEQ IDNO:23) from A. thaliana and UGT85C2 (SEQ ID NO:25) from S. rebaudiana.

The expression cassettes from these 17 plasmids are excised after anAscI+SrfI digestion, purified and then randomly concatenated in ligationreactions to create artificial yeast chromosomes (“eYACs”). From 30 to200 ug of DNA are prepared from 10 each of the cassette-containing entryvectors and the cassettes are randomly concatenated into eYACs byligation with T4 ligase in a 3 hour reaction. The success of theconcatenation reaction is assessed by the viscosity of the reactionmixture, since concatenated DNA is highly viscous. DNA fragments(“arms”) containing a centromere, two telomeres and the LEU2 and TRP1selection markers are added to the end of the 15 concatenated expressioncassettes, thereby creating functional eYACs. The eYACs are transformedinto transformation-competent spheroplasts of yeast strain erg7 byzymolyase digestion of the yeast cell wall, followed by treatment with aCaCl₂)/PEG buffer, making the spheroplasts permeable to large moleculessuch as eYACs. After transformation, the yeast spheroplasts are embeddedin a “noble agar” based solid growth medium, in which regeneration ofthe cell wall can take place. Colonies appear from 4-8 days afterinoculation. The regeneration medium lacks the amino acids leucine andtryptophan, thus selecting for the presence of double-armed eYACs in theyeast cells. One hundred transformants are selected and analyzed forproduction of mogrosides I E1, I A1 and II E, LC-MS (LiquidChromatography-coupled Mass Spectrometry (Triple Quadropole)).

Each transformant is re-streaked and tested for yeast strain markers andthe genetic presence of both arms of the eYAC, i.e., the LEU2 and TRP1markers. More than 95% of the transformants has the correct genotype.Each transformant is given a CEY designation number. Initially, 48 CEYsare grown in 50 ml of Synthetic Complete medium (SC) in 100 mlEhrlenmeyer flasks, without methionine, so as to induce gene expressionfrom the eYACs, and without tryptophan, leucine and histidine, so as tocounter-select for loss of eYACs. The cultures have a start densitycorresponding to an OD600 of 0.25, and they are inoculated for 48 h at30 C, with slow shaking (150 rpm). After 24 hours, 1 ml supernatant fromeach culture is collected and subjected to LC-MS analysis. Positive CEYs(i.e., those producing any of the mogrosides assayed for) are subjectedto PCR analysis in order to assess which CYP genes are present on theharbored eYAC and thus identifying the mogrol pathway P450 enzymes.

Example 9

Boosting Mogrol Pathway Precursor Availability

The background strain used in this study is the BY4742 strain deletedfor the TRP1 gene. This strain is called EFSC301. To increase theavailability of oxidosqualene and dioxidosqualene in this laboratoryyeast strain, the promoter of the endogenous ERG7 gene was displaced bya PCR fragment consisting of the Nurseothricin marker (NatMX) and theCUP1 cupper inducible promoter. This displacement gives lowtranscription and thereby low expression of ERG7 when the yeast strainis grown in normal growth medium like Synthetic Complete medium (SCmedium). ERG7 encode the lanosterol synthase and lowered expression isknown to result in accumulation of oxidosqualene and dioxidosqualene inbaker's yeast. Oxidosqualene is generally the precursor of triterpenoidsand possibly a precursor of the mogrol pathway. To further increaseoxidosqualene and dioxidosqualene availability the squalene epoxidaseencoded by ERG1 was overexpressed by a GPD1 promoter from a gene copyintegrated into the genome. The sequence of the squalene epoxidaseencoded by ERG1 is provided herein as SEQ ID NO:54. Furthermore atruncated copy of the yeast HMG reductase (tHMG1) was expressed from agenomically integrated gene copy, with expression from a GPD1 promoter.The resulting strain is called EFSC3027.

The successful boosting of oxidosqualene and dioxidosquale production inthe strain EFSC3027 was demonstrated by production oftetrahydroxysqualene when either one of two soluble S. grosvenoriiepoxide hydrolases was expressed in this strain. One epoxide hydrolasewas S. grosvenorii Epoxide hydrolase 1 of SEQ ID NO:38. In order toprepare yeast expressing this a S. cerevisiae codon optimized S.grosvenorii Epoxide hydrolase 1 gene sequence of SEQ ID NO:37 wasintroduced in the yeast strain EFSC3027. The other epoxide hydrolase wasS. grosvenorii Epoxide hydrolase 2 of SEQ ID NO:40. In order to prepareyeast expressing this a S. cerevisiae codon optimized S. grosvenoriiEpoxide hydrolase 1 gene sequence of SEQ ID NO:39 was introduced in theyeast strain EFSC3027. FIG. 7 shows the LC-MS mass peak 501corresponding to the proton plus Na+ adduct of tetrahydroxysqualene in asample from yeast strain EFSC3027 transformed with a plasmid expressingS. grosvenorii Epoxide hydrolase 2. Tetrahydroxysqualene is made by thehydrolysis of 2,3 and 22,23 epoxide bonds of dioxidosqualene. Noaccumulation of tetrahydroxy squalene was detected in the EFSC301background strain. Samples were made by boiling culture aliquots in 50%DMSO and then pelleting of cell material by centrifugation. Supernatantswere then measured by ESI LC-MS.

A similar system for boosting oxidosqualene availability for β-amyrinproduction was described by Kirby, J et al in FEBS Journal 275 (2008)1852-1859

Example 10

Production of Cucurbitadienol in Yeast Strain EFSC3027

When a S. cerevisiae codon optimized gene copy of the Siraitiagrosvenorii cucurbitadienol synthase of Accession No HQ128567 (sequenceprovided herein as SEQ ID NO:42) is integrated into the genome of yeaststrain EFSC3027 and transcription of this gene is driven by the GPD1promoter, the expression of the cucurbitadienol synthase results inproduction of cucurbitadienol in the yeast strain in amounts that areeasily detectable by ESI LC-MS (see FIG. 8). The amino acid sequence ofSiraitia grosvenorii cucurbitadienol synthase is provided herein as SEQID NO:43. The strain comprising SEQ ID NO:42 producing cucurbitadienolis called EFSC3498. Yeast strains were grown at 30° C. for 5 days insynthetic complete medium containing 2% glucose, and cucurbitadienol wasextracted by boiling a culture sample in 50% ethanol/20% KOH for 5minutes followed by extraction with an equal volume of hexane and thenevaporation of hexane and resuspension of dried extract in methanol.FIG. 8 shows the LC-MS chromatogram peak of lanosterol in EFSC3027(upper frame) and the LC-MS chromatogram peaks of cucurbitadienol andlanosterol in EFSC3498 (lower frame). The peak corresponding tolanosterol shows a retention time of ˜8.05 whereas the peakcorresponding to cucurbitadienol has a retention time of 7.85. Bothlanosterol and cucurbitadienol shows a mass in the LC-MS chromatogram of409.4 (proton adduct minus one H₂O)

Example 11

Production of oxo and hydroxy cucurbitadienol in S. cerevisiae When thecucurbitadienol producing yeast strain EFSC3498 (prepared as describedin Example 10) is transformed with two plasmids, one expressing the S.grosvenorii CYP5491 from a TEF1 promoter, the other expressing the S.grosvenorii CPR4497 also from a TEF1 promoter (DNA sequence encodingCPR4497 provided as SEQ ID NO:14) three conspicuous peaks emerge (seeFIG. 9 for LC-MS chromatogram peaks). The amino acid sequence of S.grosvenorii CYP5491 is provided herein as SEQ ID NO:44 and the DNAsequence encoding S. grosvenorii CYP5491 is provided as SEQ ID NO:14.The amino acid sequence of S. grosvenorii CPR4497 is provided herein asSEQ ID NO:46 and the DNA sequence encoding S. grosvenorii CPR4497 isprovided as SEQ ID NO:45. The upper frame in FIG. 9 shows the LC-MSchromatogram with the three peaks made when CYP5491 and CPR4497 areexpressed in EFSC3498, while the three lower frames show thefragmentation spectrum of these three peaks. CYP5491 is 99% identical toacc. no. HQ128570 and HQ128571 at both the amino acid and nucleotidesequence level. The masses of the 3 peaks (443.38, 441.37 and 457.36)correspond in weight to proton adducts of hydroxylated cucurbitadienol,oxo cucurbitadienol and hydroxy plus oxo cucurbitadienol respectively.Without being bound by theory it is believed that the hydroxylatedcucurbitadienol (protonated mass 443.38) and oxidated cucurbitadienol(protonated mass 441.37) is 11-hydroxy-cucurbitadienol and11-oxo-cucurbitadienol, respectively. The peak that corresponds to bothoxo plus hydroxy cucurbitadienol (protonated mass 457.36) could be11-oxo-24,25 epoxy cucurbitadienol, formed, either from cyclization ofdioxidosqualene by the cucurbitadienol synthase and 11 hydroxylation byCYP5491 (FIG. 10B) or by CYP5491 being multifunctional, making both the11-oxidation and the 24,25-epoxidation (FIG. 10A).

Example 12

Glycosylation of Mogrol in S. cerevisiae by Expression of S. grosvenoriiUGTs

UGTs 98, SK98 and 1576 were cloned from S. grosvenorii leaf and rootcDNA by primers designed from fruit gene contigs assembled from illuminasequencing data. S. grosvenorii was purchased from Horizon Herbs, LLC,United States. The DNA sequence and protein sequence of UGT98 areprovided herein as SEQ ID NO:51 and 53, respectively, whereas a SEQ IDNO:52 provides a DNA sequence encoding UGT98 codon optimised forexpression in S. cerevisiae. The DNA sequence and protein sequence ofUGTSK98 are provided herein as SEQ ID NO:49 and 50, respectively, TheDNA sequence and protein sequence of UGT1576 are provided herein as SEQID NO:47 and 48, respectively. Yeast strain EFSC1563 has a deletion ofthe EXG1 gene and of the EXG2 gene both encoding andexo-1,3-beta-Glucanase. When yeast strain EFSC1563 (EFSC301 exg1 exg2)is transformed with a plasmid expressing UGT1576 driven by a GPD1promoter and fed mogrol to a concentration in the growth medium of10-100 uM, a clear formation of mogroside I A1 is detected by LC-MS(FIG. 11B). The produced mogroside I A1 shows the same retention time asthe reference mogroside I A1 in the LC-MS analysis. FIG. 11A shows theLC-MS chromatogram of reference mogroside I A1, while 11B shows the peakfrom a sample of EFSC1563 expressing UGT1576 in a culture fed 50 uMmogrol. These data show that the UGT1576 gene encodes aglycosyltransferase with mogrol 24-OH UDP-glycosyltransferase activity.Samples were made by mixing culture aliquot 1:1 with DMSO followed byboiling (80° C.) for 5 minutes and pelleting by centrifugation.Supernatants were then subjected to ESI LC-MS.

When UGTs 98 and SK98 cloned into yeast expression plasmids withexpression from GPD1 promoters are transformed into EFSC1563 withoutco-transformation of a UGT1576 expression plasmid, no conversion of fedmogrol is detected. In contrast, co-expression of UGT98 or UGT SK98 withUGT1576 in EFSC1563 fed with mogrol results in further glycosylation ofmogroside I A1. UGT SK98 co-expressed with UGT1576 results in productionof di-glycosylated mogrol (mogroside II A, FIG. 12A), whileco-expression with UGT98 results in di and tri-glycosylated mogrol(middle and lower frames, FIG. 12B). The di-glycosylated mogrol that isformed by both UGT98 and UGT SK98 has a different retention time thanmogroside II E and mogroside II A1 during LC-MS, making it likely thatit is mogroside II A. This means that both UGT98 and UGT SK98 cancatalyse a 1,2 glucosylation of the glucose of mogroside I A1. UGT98appears to be multifunctional, catalysing 1,2 glycosylation of mogrosideI A1 resulting in production of mogroside II A, followed by what may bea 1,6 glycosylation of mogroside II A to form mogroside III A1 (FIG.12B). We believe that UGT98 catalyses 1,6 glycosylation of mogroside IIbecause mogrol itself is not glycosylated by the UGT98. It is thereforelikely that the UGT98 is multifunctional, being both a 1,2 and 1,6UDP-glucose glycosyl transferase of the 24-glucose moiety of mogrosides.UGTs 98 and SK98 belong to the UGT91 family of UDP-glucoseglycosyltransferases and members of this family are known to be 1,2 and1,6 glycosyltransferases.

FIG. 13 schematically summarizes the glycosylation reactions from mogrolto mogroside Ill A1

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A method of producing a mogrol precursor, amogroside precursor, and/or a mogroside compound in a recombinant hostcell, comprising: (a) a gene encoding a polypeptide capable ofcatalyzing conversion of oxido-squalene to produce cucurbitadienol;wherein the polypeptide comprises a polypeptide having at least 90%sequence identity to the amino acid sequence set forth in SEQ ID NO:1;(b) a gene encoding a polypeptide capable of catalyzing conversion ofdioxido-squalene to produce 24,25 epoxy cucurbitadienol; wherein thepolypeptide comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:1; (c) a geneencoding a polypeptide capable of catalyzing hydroxylation ofcucurbitadienol to produce 11-hydroxy-cucurbitadienol; wherein thepolypeptide comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence encoded by a nucleotide sequence setforth in SEQ ID NO:14; (d) a gene encoding a polypeptide capable ofcatalyzing conversion of 11-hydroxy-cucurbitadienol to produce mogrol;wherein the polypeptide comprises a polypeptide having at least 90%sequence identity to the amino acid sequence encoded by a nucleotidesequence set forth in SEQ ID NO:5; (e) a gene encoding a polypeptidecapable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol toproduce 11-hydroxy-24,25 epoxy cucurbitadienol; wherein the polypeptidecomprises a polypeptide having at least 90% sequence identity to theamino acid sequence encoded by a nucleotide sequence set forth in SEQ IDNO:5; (f) a gene encoding a polypeptide capable of catalyzing conversionof 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol; wherein thepolypeptide comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence encoded by a nucleotide sequence setforth in SEQ ID NO:5; (g) a gene encoding a polypeptide capable ofcatalyzing epoxidation of cucurbitadienol to produce 24,25 epoxycucurbitadienol; wherein the polypeptide comprises a polypeptide havingat least 90% sequence identity to the amino acid sequence encoded by anucleotide sequence set forth in SEQ ID NO:5; (h) a gene encoding apolypeptide capable of catalyzing hydroxylation of 24,25 epoxycucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;wherein the polypeptide comprises a polypeptide having at least 90%sequence identity to the amino acid sequence encoded by a nucleotidesequence set forth in SEQ ID NO:14; and/or (i) a gene encoding apolypeptide capable of beta-1,6-glycosylation of the C2′ position of the24-O-glucose and/or beta-1,2-glycosylation of the C6′ position of the3-O-glucose and/or the 24-O-glucose of the glycosylated mogrosidecompound; wherein the one or more polypeptides comprises a polypeptidehaving at least 90% sequence identity to the amino acid sequence encodedby the nucleotide sequence set forth in SEQ ID NO:26; wherein at leastone of the genes is a recombinant gene; comprising growing therecombinant host cell in a culture medium, under conditions in which thegenes are expressed; and thereby producing the mogrol precursor, themogroside precursor, and/or the mogroside compound in the recombinanthost cell.
 2. The method of claim 1, wherein the recombinant host cellcomprises genes recited in items (a), (c), (d), and (i).
 3. The methodof claim 1, wherein the recombinant host cell comprises genes recited initems (a), (c), (e), (f), and (i).
 4. The method of claim 1, wherein therecombinant host cell comprises genes recited in items (a), (g), (h),(f), and (i).
 5. The method of claim 1, wherein the recombinant hostcell comprises genes recited in items (c), (d), and (i).
 6. The methodof claim 1, wherein the recombinant host cell comprises genes recited initems (b), (f), (h), and (i).
 7. The method of claim 1, wherein therecombinant host cell comprises genes recited in items (f), (h), and(i).
 8. The method of claim 1, wherein the recombinant host cellcomprises genes recited in item (i).
 9. The method of claim 1, furthercomprising isolating the produced mogrol precursor, the mogrosideprecursor, and/or the mogroside compound.
 10. The method of claim 1,wherein the mogrol precursor is oxidosqualene.
 11. The method of claim1, wherein the mogroside precursor is a tri-glycosylated mogrol.
 12. Themethod of claim 1, wherein the mogroside compound is a mogrosidecompound di-glycosylated at C3′ position and tri-glycosylated at C24′.13. The method of claim 12, wherein the mogroside compounddi-glycosylated at C3′ position and tri-glycosylated at C24′ position ismogroside V.
 14. An in vivo method for transferring a sugar moiety to amogrol, a glycosylated mogroside compound, or both the mogrol and theglycosylated mogroside compound, comprising contacting the mogrol, theglycosylated mogroside compound, or both the mogrol and the glycosylatedmogroside compound with one or more recombinant polypeptides capable ofbeta 1,6-glycosylation of the C2′ position of the 24-O-glucose and/orbeta-1,2-glycosylation of the C6′ position of the 3-O-glucose and/or the24-O-glucose of the glycosylated mogroside compound and one or moreUDP-sugars, under suitable reaction conditions for the transfer of oneor more sugar moieties from the one or more UDP-sugars to the mogrol,the glycosylated mogroside compound, or both the mogrol and theglycosylated mogroside compound; the method comprising growing arecombinant host cell comprising a gene encoding a polypeptide capableof beta-1,6-glycosylation of the C2′ position of the 24-O-glucose and/orbeta-1,2-glycosylation of the C6′ position of the 3-O-glucose and/or the24-O-glucose of the glycosylated mogroside compound; wherein the one ormore polypeptides comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence encoded by the nucleotide sequenceset forth in SEQ ID NO:26; wherein at least one of the genes is arecombinant gene, under conditions in which one or more of the genes areexpressed; wherein contacting the mogrol, the mogroside compoundglycosylated at C3′ position and tri-glycosylated at C24′ position withthe polypeptide comprises contacting the mogroside compound glycosylatedat C3′ position and tri-glycosylated at C24′ position with thepolypeptide produced by the recombinant host cell; wherein therecombinant host cell is a microorganism that is a plant cell, amammalian cell, an insect cell, a fungal cell, an algal cell, or abacterial cell; and wherein a mogroside compound is produced in a cellculture broth upon transfer of the sugar moiety.
 15. The method of claim14, wherein the UDP-sugar is UDP-glucose, and the mogroside compounddi-glycosylated at C3′ position and tri-glycosylated at C24′ position isproduced upon transfer of the glucose moiety to the C6′ position of the3-0-glucose of the mogroside compound glycosylated at C3′ position andtri-glycosylated at C24′ position.
 16. The method of claim 14, whereinthe one or more UDP-sugar comprises UDP-glucose.
 17. The method of claim14, wherein the mogrol is a plant-derived mogrol.
 18. A method ofproducing a mogrol precursor, a mogroside precursor, and/or a mogrosidecompound, comprising whole cell bioconversion of a plant-derived or asynthetic mogrol precursor or a mogroside precursor in a cell culturemedium of a recombinant host cell comprising: (a) a gene encoding apolypeptide capable of catalyzing conversion of oxido-squalene toproduce cucurbitadienol; wherein the polypeptide comprises a polypeptidehaving at least 90% sequence identity to the amino acid sequence setforth in SEQ ID NO:1; (b) a gene encoding a polypeptide capable ofcatalyzing conversion of dioxido-squalene to produce 24,25 epoxycucurbitadienol; wherein the polypeptide comprises a polypeptide havingat least 90% sequence identity to the amino acid sequence set forth inSEQ ID NO:1; (c) a gene encoding a polypeptide capable of catalyzinghydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;wherein the polypeptide comprises a polypeptide having at least 90%sequence identity to the amino acid sequence encoded by a nucleotidesequence set forth in SEQ ID NO:14; (d) a gene encoding a polypeptidecapable of catalyzing conversion of 11-hydroxy-cucurbitadienol toproduce mogrol; wherein the polypeptide comprises a polypeptide havingat least 90% sequence identity to the amino acid sequence encoded by anucleotide sequence set forth in SEQ ID NO:5; (e) a gene encoding apolypeptide capable of catalyzing epoxidation of11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxycucurbitadienol; wherein the polypeptide comprises a polypeptide havingat least 90% sequence identity to the amino acid sequence encoded by anucleotide sequence set forth in SEQ ID NO:5; (f) a gene encoding apolypeptide capable of catalyzing conversion of 11-hydroxy-24,25 epoxycucurbitadienol to produce mogrol; wherein the polypeptide comprises apolypeptide having at least 90% sequence identity to the amino acidsequence encoded by a nucleotide sequence set forth in SEQ ID NO:5; (g)a gene encoding a polypeptide capable of catalyzing epoxidation ofcucurbitadienol to produce 24,25 epoxy cucurbitadienol; wherein thepolypeptide comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence encoded by a nucleotide sequence setforth in SEQ ID NO:5; (h) a gene encoding a polypeptide capable ofcatalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce11-hydroxy-24,25 epoxy cucurbitadienol; wherein the polypeptidecomprises a polypeptide having at least 90% sequence identity to theamino acid sequence encoded by a nucleotide sequence set forth in SEQ IDNO:14; and/or (i) a gene encoding a polypeptide capable of beta1,6-glycosylation of the C2′ position of the 24-O-glucose, and/orbeta-1,2-glycosilation of the C6′ position of the 3-O-glucose and/or the24-O-glucose of the glycosylated mogroside compound; wherein the one ormore polypeptides comprises a polypeptide having at least 90% sequenceidentity to the amino acid sequence encoded by the nucleotide sequenceset forth in SEQ ID NO:26; wherein at least one of the genes is arecombinant gene; wherein at least one of the polypeptides is arecombinant polypeptide expressed in the recombinant host cell; whereinthe recombinant host cell is a microorganism that is a plant cell, amammalian cell, an insect cell, a fungal cell, an algal cell, or abacterial cell; and producing the mogrol precursor, the mogrosideprecursor, and/or the mogroside compound thereby.
 19. The method ofclaim 1, wherein the recombinant host cell is a microorganism that is aplant cell, a mammalian cell, an insect cell, a fungal cell, an algalcell, or a bacterial cell.